Simultaneous detection of expression of five genes in a whole-mount Drosophila embryo by fluorescence in situ hybridization (FISH) with five RNA probes. Red: sog labeled using aminoallyl UTP (Product # A21663, A32765) and Alexa Fluor® 647 succinimidyl ester (Product # A-20006, A20106). Green: ind labeled with DNP, followed by rabbit anti-dinitrophenyl-KLH IgG antibody (Product # A-6430) prelabeled with the ... View More

Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody in Immunocytochemistry (ICC/IF)

Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody in Flow Cytometry (Flow)

Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody

Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody in Immunohistochemistry, Immunocytochemistry (IHC, ICC/IF)

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Localization of TBC protein candidates in the parasite.Immunofluorescence assays (IFAs) of endogenously epitope-tagged TBC1-10, 12-18 parasites were performed for parasites grown for 24 h. a Confocal co-localization of the epitope Ty fused at TBC1, 5, 6, 9, and 14 (green) with the ER marker BIP (red). b Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent region of TBC1, 5, 6, 9, and 14 with BIP were analyzed using the colocalization and ROI intensity analysis module in NIS software. c Confocal co-localization of the epitope Ty fused at TBC2, 3, and 18 (green) with the TGN marker STX6 (red) and the cis-Golgi marker GRASP (red). d Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent foci of TBC2, 3, and 18 with STX6 and GRASP. e TBC4, 8, 12, 13, 16, and 17 are localized to the vesicles of the cytosol and TBC10 are localized to the cytosol and the PV (arrowhead). The IFA analyses were achieved using anti-Ty antibodies (green), and the outline of the parasites was visualized using anti-GAP45 antibodies (red). f Confocal co-localization of the epitope Ty fused at TBC15 (green) with the IMC marker IMC1 (red). Data are presented as mean ± SD (n = 50 parasites), with the Mann-Whitney test used for the PCC of TBC2 and 3, and the unpaired t-test used for the PCC. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Localization of TBC protein candidates in the parasite.Immunofluorescence assays (IFAs) of endogenously epitope-tagged TBC1-10, 12-18 parasites were performed for parasites grown for 24 h. a Confocal co-localization of the epitope Ty fused at TBC1, 5, 6, 9, and 14 (green) with the ER marker BIP (red). b Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent region of TBC1, 5, 6, 9, and 14 with BIP were analyzed using the colocalization and ROI intensity analysis module in NIS software. c Confocal co-localization of the epitope Ty fused at TBC2, 3, and 18 (green) with the TGN marker STX6 (red) and the cis-Golgi marker GRASP (red). d Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent foci of TBC2, 3, and 18 with STX6 and GRASP. e TBC4, 8, 12, 13, 16, and 17 are localized to the vesicles of the cytosol and TBC10 are localized to the cytosol and the PV (arrowhead). The IFA analyses were achieved using anti-Ty antibodies (green), and the outline of the parasites was visualized using anti-GAP45 antibodies (red). f Confocal co-localization of the epitope Ty fused at TBC15 (green) with the IMC marker IMC1 (red). Data are presented as mean ± SD (n = 50 parasites), with the Mann-Whitney test used for the PCC of TBC2 and 3, and the unpaired t-test used for the PCC. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Localization of TBC protein candidates in the parasite.Immunofluorescence assays (IFAs) of endogenously epitope-tagged TBC1-10, 12-18 parasites were performed for parasites grown for 24 h. a Confocal co-localization of the epitope Ty fused at TBC1, 5, 6, 9, and 14 (green) with the ER marker BIP (red). b Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent region of TBC1, 5, 6, 9, and 14 with BIP were analyzed using the colocalization and ROI intensity analysis module in NIS software. c Confocal co-localization of the epitope Ty fused at TBC2, 3, and 18 (green) with the TGN marker STX6 (red) and the cis-Golgi marker GRASP (red). d Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent foci of TBC2, 3, and 18 with STX6 and GRASP. e TBC4, 8, 12, 13, 16, and 17 are localized to the vesicles of the cytosol and TBC10 are localized to the cytosol and the PV (arrowhead). The IFA analyses were achieved using anti-Ty antibodies (green), and the outline of the parasites was visualized using anti-GAP45 antibodies (red). f Confocal co-localization of the epitope Ty fused at TBC15 (green) with the IMC marker IMC1 (red). Data are presented as mean ± SD (n = 50 parasites), with the Mann-Whitney test used for the PCC of TBC2 and 3, and the unpaired t-test used for the PCC. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Localization of TBC protein candidates in the parasite.Immunofluorescence assays (IFAs) of endogenously epitope-tagged TBC1-10, 12-18 parasites were performed for parasites grown for 24 h. a Confocal co-localization of the epitope Ty fused at TBC1, 5, 6, 9, and 14 (green) with the ER marker BIP (red). b Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent region of TBC1, 5, 6, 9, and 14 with BIP were analyzed using the colocalization and ROI intensity analysis module in NIS software. c Confocal co-localization of the epitope Ty fused at TBC2, 3, and 18 (green) with the TGN marker STX6 (red) and the cis-Golgi marker GRASP (red). d Statistics of Pearson correlation coefficient (PCC). The PCC values over the merged fluorescent foci of TBC2, 3, and 18 with STX6 and GRASP. e TBC4, 8, 12, 13, 16, and 17 are localized to the vesicles of the cytosol and TBC10 are localized to the cytosol and the PV (arrowhead). The IFA analyses were achieved using anti-Ty antibodies (green), and the outline of the parasites was visualized using anti-GAP45 antibodies (red). f Confocal co-localization of the epitope Ty fused at TBC15 (green) with the IMC marker IMC1 (red). Data are presented as mean ± SD (n = 50 parasites), with the Mann-Whitney test used for the PCC of TBC2 and 3, and the unpaired t-test used for the PCC. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


TBC9 is essential for parasite replication.a Schematic diagram for generation of the TBC9-mAID fusion by tagging the gene at the C-terminus with mAID-3xHA using a CRISPR/Cas9 approach in the parental line RHΔku80Δhxgprt/TIR1. The homologous regions (HR) were retrieved from the upstream of the stop codon and the downstream of the 3’ sgRNA targeting site, while the HXGPRT represents the resistant expression cassette. b The TBC9-mAID-3xHA parasite line was grown in IAA or ethanol for 24 h, followed by fixation for IFA analyses, using antibodies against MLC1 (red) and the epitope tag HA (green). Scale bar = 5 µm. c Western blot analysis for the TBC9-mAID-3xHA line grown for induction of auxin for different times (hrs) as indicated. Actin served as a load control. d Plaque formation of the TIR1 and TBC9-mAID lines. Parasites were grown on HFF monolayers in ± IAA, followed by fixation and staining with crystal violet after 7 days of culturing. Scale bar = 2 mm. e Parasite replication of the TIR1 and TBC9-mAID lines. Parasites were grown in ±IAA for 24 h, followed by IFA analysis of parasites by antibodies against GAP45. Vacuoles with different parasite numbers (1, 2, 4, 8, and ≥16) were counted (≥200 vacuoles for each replicate). Data (N = 3 independent experiments; n = 3 replicates) were shown with means &. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


The dual-finger catalytic sites are essential for the activity of TBC9 in the parasite.a-c IFA detection of TBC9-mAID-3xHA line complemented with TBC9wt-2TY (TBC9wt). Parasites were grown on HFF monolayers for 16 h, followed by ± IAA treatment for 13 h before processing for IFA analyses using antibodies against the proteins indicated in the images: MLC1, GAP45, and IMC1 for the inner membrane complex; Tubulin for the subpellicular microtubules76; HSP60 for the mitochondrion, and IPPS for the ER. Scale bar = 5 µm. d Diagram illustrating the TBC dual-finger catalytic sites of TBC9. The arginine and glutamine in the sites were mutated into alanine, respectively. Yellow boxes depict arginine (R finger) and glutamine (Q finger), red boxes depict residues that were mutated to alanine (A). e IFA detection of the TBC9-mAID-3xHA line complemented with TBC9R74A-2TY or TBC9Q101A-2TY. Parasites were grown on HFF monolayers for 16 h, followed by ± IAA treatment for 13 h before processing for IFA analyses using antibodies against the TY (green) and MLC1 (red). Hoechst (blue) dye was used to stain the nucleus. Scale bar = 5 µm. f Parasite replication of the TBC9-mAID, TBC9wt, TBC9R74A and TBC9Q101A lines. Vacuoles with different parasite numbers (1, 2, 4, 8, and ≥16) were counted (≥200 vacuoles for each replicate). Two-way ANOV. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Depletion of TBC9 affects the early vesicle transport pathway and its related proteins in T. gondii.Immunofluorescence analysis of normal or mis-localization of protein sorting pathway proteins and organelle marker proteins upon depletion of TBC9 in T. gondii. TBC9-mAID parasites were grown on HFF monolayers for 16 h, followed by ± IAA treatment for 13 h before processing for IFA analyses using the corresponding antibodies. MLC1 or IMC1 (red) for the inner membrane complex, and Hoechst (blue) dye was used to stain the nucleus. a BIP and IPPS staining for the ER. b Vacuoles with normal localization or mis-localization of ER were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Tukey’s multiple comparison test was performed. c GRASP was tagged with Ty using a CRISPR approach in the TBC9-mAID line. Six distinct types of Golgi were found for the GRASP staining. d Vacuoles with different GRASP types were specifically counted during either daughter IMC formation or only mother IMC (≥200 vacuoles for each replicate). Two-way ANOVA with Sidak’s multiple comparison test was performed. e Staining of SORTLR and VPS35 tagged with Ty in TBC9-mAID line. f Vacuoles with normal localization or mis-localization of SORTLR or VPS35 were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Tukey’s multiple . Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Depletion of TBC9 affects the early vesicle transport pathway and its related proteins in T. gondii.Immunofluorescence analysis of normal or mis-localization of protein sorting pathway proteins and organelle marker proteins upon depletion of TBC9 in T. gondii. TBC9-mAID parasites were grown on HFF monolayers for 16 h, followed by ± IAA treatment for 13 h before processing for IFA analyses using the corresponding antibodies. MLC1 or IMC1 (red) for the inner membrane complex, and Hoechst (blue) dye was used to stain the nucleus. a BIP and IPPS staining for the ER. b Vacuoles with normal localization or mis-localization of ER were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Tukey’s multiple comparison test was performed. c GRASP was tagged with Ty using a CRISPR approach in the TBC9-mAID line. Six distinct types of Golgi were found for the GRASP staining. d Vacuoles with different GRASP types were specifically counted during either daughter IMC formation or only mother IMC (≥200 vacuoles for each replicate). Two-way ANOVA with Sidak’s multiple comparison test was performed. e Staining of SORTLR and VPS35 tagged with Ty in TBC9-mAID line. f Vacuoles with normal localization or mis-localization of SORTLR or VPS35 were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Tukey’s multiple . Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Depletion of TBC9 affects the early vesicle transport pathway and its related proteins in T. gondii.Immunofluorescence analysis of normal or mis-localization of protein sorting pathway proteins and organelle marker proteins upon depletion of TBC9 in T. gondii. TBC9-mAID parasites were grown on HFF monolayers for 16 h, followed by ± IAA treatment for 13 h before processing for IFA analyses using the corresponding antibodies. MLC1 or IMC1 (red) for the inner membrane complex, and Hoechst (blue) dye was used to stain the nucleus. a BIP and IPPS staining for the ER. b Vacuoles with normal localization or mis-localization of ER were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Tukey’s multiple comparison test was performed. c GRASP was tagged with Ty using a CRISPR approach in the TBC9-mAID line. Six distinct types of Golgi were found for the GRASP staining. d Vacuoles with different GRASP types were specifically counted during either daughter IMC formation or only mother IMC (≥200 vacuoles for each replicate). Two-way ANOVA with Sidak’s multiple comparison test was performed. e Staining of SORTLR and VPS35 tagged with Ty in TBC9-mAID line. f Vacuoles with normal localization or mis-localization of SORTLR or VPS35 were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Tukey’s multiple . Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Depletion of the TBC9 affects daughter bud formation and organelles duplication.a, b TBC9-mAID parasites were grown on HFF monolayers for 16 h, followed by ± IAA treatment for 13 h before processing for IFA analyses. Hoechst (blue) dye was used to stain the nucleus. Using antibody combinations against IMC1 (red) and GAP45 (green), IFA results were obtained for morphologically normal parasites without daughter bud formation (a, white arrowheads) and morphologically fragmented parasites with daughter bud formation (b, green arrowheads), which were classified into four types (type A-D) by daughter formation and the number of nucleus (yellow arrowheads). c Vacuoles with 4 types in (A-D) were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Sidak’s multiple comparison test was performed. d TBC9-mAID parasites were grown for 6 h, followed by ± IAA treatment for 16 h before processing for IFA analysis. Antibodies against IMC1 (green) and GAP45 (red) were used for staining the inner membrane complex. e Vacuoles with 4 types (the same as in a, b) in (d) were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Sidak’s multiple comparison test was performed. f Staining of Ty-tagged GRASP in the TBC9-mAID line, and the six distinct types of Golgi are consistent with those shown in Fig. 5c. g Vacuoles. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Depletion of the TBC9 affects daughter bud formation and organelles duplication.a, b TBC9-mAID parasites were grown on HFF monolayers for 16 h, followed by ± IAA treatment for 13 h before processing for IFA analyses. Hoechst (blue) dye was used to stain the nucleus. Using antibody combinations against IMC1 (red) and GAP45 (green), IFA results were obtained for morphologically normal parasites without daughter bud formation (a, white arrowheads) and morphologically fragmented parasites with daughter bud formation (b, green arrowheads), which were classified into four types (type A-D) by daughter formation and the number of nucleus (yellow arrowheads). c Vacuoles with 4 types in (A-D) were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Sidak’s multiple comparison test was performed. d TBC9-mAID parasites were grown for 6 h, followed by ± IAA treatment for 16 h before processing for IFA analysis. Antibodies against IMC1 (green) and GAP45 (red) were used for staining the inner membrane complex. e Vacuoles with 4 types (the same as in a, b) in (d) were counted (≥200 vacuoles for each replicate). Two-way ANOVA with Sidak’s multiple comparison test was performed. f Staining of Ty-tagged GRASP in the TBC9-mAID line, and the six distinct types of Golgi are consistent with those shown in Fig. 5c. g Vacuoles. Image collected and cropped by CiteAb under a CC-BY license from the following publication: TBC9, an essential TBC-doma… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Activation of human brain microvascular endothelial cells (HBMECs) cultured in DMEM supplemented with extracellular vesicle-depleted fetal bovine serum (EVdS). HBMECs were cultured in 25 cm2 (T25) flasks with DMEM supplemented with 10% FBS or TNF-α (10 ng/mL). Afterward, the cells were suspended in sterile PBS, labeled with Annexin-V conjugated to phycoerythrin and propidium iodide (PI), and evaluated by flow cytometry (A-C). Annexin-V+/PI− (early apoptotic cells) (B) and annexin-V+/PI+ (late apoptotic cells) (C) were graphically plotted. RNA from another cultured set of HBMEC was isolated for cDNA synthesis and quantification of IL6, IL8, and VEGF by qPCR (D-F). Intercellular adhesion molecule 1 (ICAM-1) expression was evaluated by staining HBMEC cultured in the absence of FBS (0%), 10% FBS, or EV-depleted FBS (EVdS) for 2 or 24 h. The cells were incubated with a primary anti-ICAM-1 antibody and a secondary anti-mouse conjugated to Alexa Fluor™ 488 (green), stained with DAPI (blue), and evaluated under a fluorescence microscope (G). The obtained images were evaluated using specific software, and the results are expressed as the integrated density (H). The data were obtained from three biological replicates and are expressed as the mean ± standard deviation (SD) (ANOVA/Tukey (B-F); Kruskal-Wallis/Dunn (H)); *: p < 0.05, **:. Image collected and cropped by CiteAb under a CC-BY license from the following publication: Unraveli… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


The expression of intercellular adhesion molecule 1 (ICAM-1) on human brain microvascular endothelial cells (HBMECs) is modulated by extracellular vesicles (EVs). EVs were obtained from HBMEC cultured in DMEM supplemented with EV-depleted FBS (EVdS) by differential centrifugation. HBMEC were grown in 24-well plates and incubated with EV2h, EV24h (100 ng/mL), TNF (10 ng/mL), or PBS for 24 h. A second experiment was conducted with cells incubated with extracellular vesicles isolated from cells cultured for 2 h (EV2h) or EV24h at concentrations of 0.01, 0.1, or 1.0 µg/mL. Following the incubation, both groups of cells were washed, fixed with 4% paraformaldehyde, and then incubated with a primary anti-ICAM-1 antibody. Subsequently, a secondary anti-mouse IgG antibody (H+L) conjugated to Alexa Fluor® 488 was applied (green), and the cells were stained with DAPI (blue). The cells were then evaluated under a fluorescence microscope (A). The obtained images were analyzed using specific software, and comparisons between EV2h and EV24h (B) and between different concentrations of EV2h (C) or EV24h (D) are expressed as the integrated density. The data were obtained from three biological replicates with five technical replicates each and are presented as the mean ± standard deviation (ANOVA/Tukey). Statistical significance was determined as fol. Image collected and cropped by CiteAb under a CC-BY license from the following publication: Unraveling the Impact o… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Application of super-resolution proximity labeling to map p-body proteome.a Schematic illustration of processing body (PB) proteome profiling via proximity labeling and a list of bait with schematics of bait-APEX2/TurboID construct. b Representative immunofluorescence (IF) images of expressed APEX2/TurboID conjugated baits and biotin-labeled patterns. Scale bar = 10 µm. c Volcano plot of differentially expressed proteins identified from DCP1a-APEX2 and NES-APEX2 construct. PB candidate proteins are marked green, and gene names of known PB proteins are labeled. d Identification overlaps of identified PB candidates among different bait-APEX2/TurboID constructs and published results based on BioID. e Network analysis of identified PB protein candidates. Listed core PB proteins (right) are marked red. f Gene Ontology analysis of identified PB protein candidates and their network using Clue-go analysis. g Representative IF images of UBAP2L localization to PB under various conditions. Arsenite was treated for 30 min. Scale bar = 5 µm. Image collected and cropped by CiteAb under a CC-BY license from the following publication: Super-resolution proximity labeling with enhanced direct identification of biotinylation sites. Commun Biol (2024) Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


MYADM is required for infection of primary intestinal organoids.a Wild type (WT) and two MYADM knockout human colonoid lines (a^+MYADM #11 and a^+MYADM #22) were infected with PeV-A1 or K26GFP (HSV-GFP) and stained for double-stranded RNA or GFP, respectively. Uninfected colonoids (Mock) served as negative controls. Right panels illustrating increased magnification of square boxes in the left panel. b Quantification of PeV-A1 infection in colonoids using RT-qPCR and plaque assay over time. Data are presented as mean valuesaEUR%0+/a^'aEUR%0SEM, naEUR%0=aEUR%03 or 4 biologically independent samples. c Quantification of K26GFP (HSV-GFP) infection in colonoids using RT-qPCR and plaque assay over time. Data are presented as mean valuesaEUR%0+/a^'aEUR%0SEM, naEUR%0=aEUR%03 biologically independent samples. Source data are provided as a Source Data file. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: MYADM binds human parechovirus 1 and is essential for viral entry. Nat Commun (2024) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/38658526), licensed under a CC BY license.


Arl4A binding modulates the association of VPS36 with MVBs.a HeLa cells were transfected for 24 h with the indicated plasmid. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and DAPI (blue). Scale bar, 10 µm. Quantification of VPS36-Myc and CD63 colocalization is shown as the Pearson coefficient. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 84 cells, VPS36-A6 = 104 cells). b HeLa cells were co-transfected with VPS36-WT-Myc or VPS36-A6-Myc and GFP-Rab5A Q79L for 24 h. The cells were fixed and then stained with anti-Myc (red) and anti-GFP (green) antibodies. The images were acquired using the Leica TCS SP8 STED microscope. Scale bar, 5 µm. Imaris software was used to quantify the 3D-colocalization coefficient values of VPS36 and Rab5 Q79L. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 16 cells, VPS36-A6 = 22 cells). c HeLa cells stably expressing control shRNA or VPS4A and VPS4B shRNA using shRNA-expressing lentivirus. After puromycin selection, the cells were transfected with the indicated plasmids. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and D - Image collected and cropped by CiteAb under a CC-BY … Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/38030597), licensed under a CC BY license.


Arl4A affects the association of VPS36 and ESCRT-III components.a HeLa cells were transfected with the indicated VPS36 plasmids and GFP-CHMP2A for 24 h and stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (red) and DAPI (blue). Scale bar, 10 µm. Quantification of VPS36-Myc and GFP-CHMP2A colocalization was obtained from three biological repeats and shown as the colocalization coefficient by ZEN software. The results represent the mean ± SD and the p-value was assessed by a two-sided t-test (VPS36-WT = 62 cells, VPS36-A6 = 74 cells). b Comparison of VPS36-WT and VPS36-A6 binding ability toward ESCRT-III component CHMP2A in vivo. HeLa cells transfected with indicated plasmids were treated with 1 mM dithiobis(succinimidyl propionate) (DSP) for 2 h and quenched by Tris-HCl before cell lysis and immunoprecipitation with GFP-Trap. The bound proteins were analyzed by western blotting with anti-Myc or anti-GFP antibodies. The amounts of coimmunoprecipitated VPS36 were determined by densitometric quantification from four experiments as indicated. The results represent the mean ± SD and the p-value was assessed by a two-sided t-test. c Co-IP of VPS36 and CHMP2A in Arl4-knockdown HeLa cells. Cells transfected with the control or Arl4A siRNA as well as the indicated plasmids were treated with 1 mM DSP for 1 h and immunoprecipited with GFP-Trap. The bound pro - Image collected and cropped by CiteAb under a CC-BY … Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/38030597), licensed under a CC BY license.


Arl4A binding modulates the association of VPS36 with MVBs.a HeLa cells were transfected for 24 h with the indicated plasmid. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and DAPI (blue). Scale bar, 10 µm. Quantification of VPS36-Myc and CD63 colocalization is shown as the Pearson coefficient. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 84 cells, VPS36-A6 = 104 cells). b HeLa cells were co-transfected with VPS36-WT-Myc or VPS36-A6-Myc and GFP-Rab5A Q79L for 24 h. The cells were fixed and then stained with anti-Myc (red) and anti-GFP (green) antibodies. The images were acquired using the Leica TCS SP8 STED microscope. Scale bar, 5 µm. Imaris software was used to quantify the 3D-colocalization coefficient values of VPS36 and Rab5 Q79L. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 16 cells, VPS36-A6 = 22 cells). c HeLa cells stably expressing control shRNA or VPS4A and VPS4B shRNA using shRNA-expressing lentivirus. After puromycin selection, the cells were transfected with the indicated plasmids. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and D - Image collected and cropped by CiteAb under a CC-BY … Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/38030597), licensed under a CC BY license.


Arl4A affects the level of EGFR ubiquitination and sorting of EGFR into MVBs.a HeLa cells were transfected with empty vector (EV) or Arl4A WT in combination with EGFR-Myc (b) C33-A cells were transfected with either control or Arl4A siRNA in combination with EGFR-Myc. a and b After 24 h of transfection with plasmids or 48 h of transfection with siRNAs, the cells were starved and stimulated with EGF (100 ng/ml) for the indicated time. Cell lysates were immunoprecipitated with Myc-Trap for EGFR-Myc and analyzed by Western blotting with antibodies against ubiquitin or Myc. Total cell lysates (input) were immunoblotted with the indicated antibodies. The amounts of ubiquitin-EGFR were determined by ImageJ software. The results represent the mean ± SD of three independent experiments and the p-values were assessed by a two-sided t-test. c HeLa cells were transfected with either empty vector (EV) or Arl4A and GFP-Rab5A Q79L for 24 h. After serum starvation, the cells were incubated with 100 ng/ml EGF for 10 min at 37 °C and then stained with anti-EGFR (red) and anti-Arl4A (blue) antibodies. Images were acquired using a Leica TCS SP8 STED microscope. Scale bar, 5 µm. Imaris 3D visualization software was used to create an enlarged endosome in a 3D orthoslicer view. Endosomes with diameters >2 µm were identified for analysis. Using the Rab5-Q79L outline to - Image collected and cropped by CiteAb under a CC-BY … Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/38030597), licensed under a CC BY license.


Co-localization of AID with UBN1 in DT40ΨV KO cells. Cells were fixed and double stained with monoclonal anti-AID (green) and polyclonal anti-UBN1 (red) antibodies. AID is predominantly located in the cytoplasm, whereas UBN1 is mostly inside the nucleus. Yellow and orange spots are produced due to the co-localization of AID with UBN1. Image collected and cropped by CiteAb under a CC-BY license from the following publication: Activation-induced cytidine deaminase an antibody diversification enzyme interacts with chromatin modifier UBN1 in B-cells. Sci Rep (2023) Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Co-localization of AID with UBN1 in Raji cells. Cells were fixed and double stained with monoclonal anti-AID (green) and polyclonal anti-UBN1 (red) antibodies. AID is predominantly located in the cytoplasm, whereas UBN1 is mostly inside the nucleus. Yellow and orange spots are produced due to the co-localization of AID with UBN1. Image collected and cropped by CiteAb under a CC-BY license from the following publication: Activation-induced cytidine deaminase an antibody diversification enzyme interacts with chromatin modifier UBN1 in B-cells. Sci Rep (2023) Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Spn infection disrupts the structural integrity of the Golgi apparatus. BEAS-2B cells were infected with Spn D39, MOI 1 for 16 h or left uninfected. Cells were fixed and fluorescence stained for Golgin-97 and the nucleus (DAPI). Binary images were generated and used to quantify the Golgi area. A Immunofluorescence images. Overlays were generated using the false-colored DAPI- and Golgin-97 images. B Quantification of the Golgi surface area, normalized to uninfected controls. (Statistics: paired two-tailed t-test; * = p < 0.05; Scale: 10 µm; at least 600 cells from 5 independent experiments were quantified) - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner. Cell Commun Signal (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37592354), licensed under a CC BY license.


Deletion of the pneumococcal peroxidase rescues Golgi integrity. BEAS-2B cells were infected with Spn D39 WT or Spn D39 ΔspxB or left untreated. Cells were fixed and fluorescence stained for Golgin-97 and the nucleus (DAPI). A Immunofluorescence images of infected and uninfected cells. B Quantification of the Golgi surface area, relative to uninfected controls. C 16 h post infection, mitochondrial activity was assessed by MTT assay. (Statistics: paired two-tailed t-test for ΔspxB vs. WT; * = p < 0.05; Scale: 10 µm; at least 500 cells from 4 independent experiments were quantified) - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner. Cell Commun Signal (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37592354), licensed under a CC BY license.


H2O2 production induces Golgi disruption. A BEAS-2B cells were infected with Spn D39 WT, MOI 1 for 16 h with or without addition of recombinant Catalase or left untreated. Cells were fixed and Golgin-97 and the nucleus stained. B Quantification of (A). C Cells were treated with H2O2 at indicated concentrations for 16 h. Cells were fixed and Golgin-97 and the nucleus stained. D Quantification of (C). (Statistics: paired two-tailed t-test; * = p < 0.05; Scale: 10 µm; at least 500 cells from 4 independent experiments were quantified) - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner. Cell Commun Signal (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37592354), licensed under a CC BY license.


H2O2 production induces Golgi disruption. A BEAS-2B cells were infected with Spn D39 WT, MOI 1 for 16 h with or without addition of recombinant Catalase or left untreated. Cells were fixed and Golgin-97 and the nucleus stained. B Quantification of (A). C Cells were treated with H2O2 at indicated concentrations for 16 h. Cells were fixed and Golgin-97 and the nucleus stained. D Quantification of (C). (Statistics: paired two-tailed t-test; * = p < 0.05; Scale: 10 µm; at least 500 cells from 4 independent experiments were quantified) - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner. Cell Commun Signal (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37592354), licensed under a CC BY license.


CSE-treated bronchospheres have decreased spheroid area and increased MUC5AC secretion in the lumen compared to control.A) Schematic of a well within a 384-well plate with bronchosphere organoids in 2.5D culture with a 3T3 feeder cell layer at the well bottom. B) Schematic of the experimental workflow. Bronchospheres were seeded and differentiated over two weeks, after which they were exposed to 0 or 3% CSE for 1-, 2-, and 3-week continuous treatment. At each timepoint, spheroids were imaged live to assess changes in size, then fixed and stained for secreted mucins. C) Representative images of live bronchospheres stained with TMRM dye that show changes in area with CSE treatment. D) Bar plot of spheroid area at all timepoints treated with 0 or 3% CSE. Conditions are comprised of 3 biological replicates (3 separate experiments) from 3 independent donors (9 experiments total). Datapoints were normalized to the average of the control condition for each donor and at each timepoint, due to donor-to-donor variability. Data is represented as mean A+- SEM and each timepoint was analyzed by paired two-tailed t-test, **p<0.01, ***p<0.001. E) Representative images of bronchospheres stained for MUC5AC and MUC5B that show increased MUC5AC secretion in the bronchospheres lumen with CSE treatment. F) Bar plot of ratio of MUC5AC and G) MUC5B cluster area to total - Image collected and cropped by CiteAb under a CC-BY license from the follow… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37384771), licensed under a CC BY license.


Attenuation of spheroid size decrease or MUC5AC increase by hit compounds.Final hit compounds (with hit concentration and corresponding target) that A) increased bronchosphere size compared to DMSO + 3% CSE control bronchospheres or B) decreased secreted MUC5AC. Mucus reduction hit compounds were further refined by manually comparing TMRM live dye-stained images of compound treated vs. control bronchospheres to observe aberrant phenotype that could indicate an unhealthy state. These images are shown in S5 Fig. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: A high-throughput cigarette smoke-treated bronchosphere model for disease-relevant phenotypic compound screening. PLoS One (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37384771), licensed under a CC BY license.


Reactivation of silenced tumor suppressor genes by CRISPRa correlates with phenotypic reprogramming in Hep3B HCC cells. A, B Transcriptional reactivation of four silenced tumor suppressor genes (TSGs), HHIP, MT1M, PZP, and TTC36, targeted individually and simultaneously (MIX 4 genes) by SpdCas9-VPR stably expressed along with the corresponding TSG-gRNA in Hep3B HCC cells. A Data shown as fold log10 change in TSG mRNA levels, evaluated by qRT-PCR, relative to SpdCas9-VPR with no gRNA (NO G). Data presented as meansaEUR%0A+-aEUR%0SEM (naEUR%0=aEUR%03) and P values were determined by one-way ANOVA with Dunnett's multiple comparisons test (****PaEUR%0


Reactivation of silenced tumor suppressor genes by CRISPRa correlates with phenotypic reprogramming in Hep3B HCC cells. A, B Transcriptional reactivation of four silenced tumor suppressor genes (TSGs), HHIP, MT1M, PZP, and TTC36, targeted individually and simultaneously (MIX 4 genes) by SpdCas9-VPR stably expressed along with the corresponding TSG-gRNA in Hep3B HCC cells. A Data shown as fold log10 change in TSG mRNA levels, evaluated by qRT-PCR, relative to SpdCas9-VPR with no gRNA (NO G). Data presented as meansaEUR%0A+-aEUR%0SEM (naEUR%0=aEUR%03) and P values were determined by one-way ANOVA with Dunnett's multiple comparisons test (****PaEUR%0


Reactivation of silenced tumor suppressor genes by CRISPRa correlates with phenotypic reprogramming in Hep3B HCC cells. A, B Transcriptional reactivation of four silenced tumor suppressor genes (TSGs), HHIP, MT1M, PZP, and TTC36, targeted individually and simultaneously (MIX 4 genes) by SpdCas9-VPR stably expressed along with the corresponding TSG-gRNA in Hep3B HCC cells. A Data shown as fold log10 change in TSG mRNA levels, evaluated by qRT-PCR, relative to SpdCas9-VPR with no gRNA (NO G). Data presented as meansaEUR%0A+-aEUR%0SEM (naEUR%0=aEUR%03) and P values were determined by one-way ANOVA with Dunnett's multiple comparisons test (****PaEUR%0


Reactivation of silenced tumor suppressor genes by CRISPRa correlates with phenotypic reprogramming in Hep3B HCC cells. A, B Transcriptional reactivation of four silenced tumor suppressor genes (TSGs), HHIP, MT1M, PZP, and TTC36, targeted individually and simultaneously (MIX 4 genes) by SpdCas9-VPR stably expressed along with the corresponding TSG-gRNA in Hep3B HCC cells. A Data shown as fold log10 change in TSG mRNA levels, evaluated by qRT-PCR, relative to SpdCas9-VPR with no gRNA (NO G). Data presented as meansaEUR%0A+-aEUR%0SEM (naEUR%0=aEUR%03) and P values were determined by one-way ANOVA with Dunnett's multiple comparisons test (****PaEUR%0


a Immunofluorescence analysis showed approximately similar biomarker expression of hLMSCs in SM for ocular surface marker (Pax6+), stem-cell biomarkers (ABCG2+, P63α+, ABCB5+) and the mesenchymal biomarkers (VIM+, CD90+, CD105+, CD 34−, HLA-DR− and CD45−) with respect to the cells in control medium; b the expression of MSC markers in hLMSCs grown in both the media was quantified using flow cytometry. More than 97% of cells were positive for CD105, CD90, and CD73, whereas less than 1% showed expression for negative markers CD45 and HLA-DR and approx. 6% of total cells were positive for CD34; c graphical representation of flow cytometry data. Blue: DAPI; Scale: 50 µM; Magnification: × 20 (all other micrographs) and 20 µM (CD73 of DMEM/F12 with 2% FBS; × 40 magnification) - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications. Stem Cell Res Ther (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37061739), licensed under a CC BY license.


Increased contribution of L-type VGCCs in SNc-DA neurons of G2019S mice but not cNurr1 mice.A Representative images of neurons in the SNc from double FISH experiments of CACNA1D mRNA, which encodes the Cav1.3 L-type channel, and TH mRNA; scale bars, 15 µm. Quantification of fluorescence for CACNA1D mRNA in individual TH-containing cells in the SNc. n = 186, 185, 135, 126 neurons from N = 3 Ctrl, 3 cNurr1, 3 WT and 3 G2019S mice. ####P < 0.0001; Mann-Whitney U-test. B Schematic representation depicting the SNc and a DA neuron. Isradipine (Isra) was used to block Cav1.3 L-type channels. C, D Representative traces of pacemaker firing recorded in the cell-attached mode in SNc-DA neurons from Ctrl, cNurr1, WT and G2019S mice before (Pre-Isra) and during (Isra) the perfusion with isradipine (0.2 µM) in control condition (aCSF, C), in slices incubated with the LRRK2 kinase inhibitor GSK2578215A (1 µM, D) or with kaempferol (KF, 5 µM, D). Graphs show the firing frequency of individual SNc-DA neurons before (Pre-Isra) and during (Isra) the perfusion with Isradipine. Cells recorded in aCSF: n = 8, 9, 13, 13 neurons from N = 6 Ctrl, 8 cNurr1, 4 WT and 5 G2019S mice (C). *P < 0.05; Paired Student’s t-test. Cells recorded in GSK2578215A: n = 6 and 6 neurons from N = 2 WT and 4 G2019S mice. Cells recorded in kaempferol: n = 10 and 11 neurons from N = 5 WT and - Image collected and cropped by CiteAb under a C… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37029193), licensed under a CC BY license.


Alcohol augments hepatic ER-mitochondria interaction.a Graphical depiction of alcohol feeding model in mice. b Mitochondria and ER morphology in the perivenous region of control diet (CD) and EtOH diet (ED)-fed mice liver sections were visualized using TEM (Scale bars, 5aEUR%0Aum). The ER and mitochondria in the TEM images were reconstructed graphically to visualize MAM formation. (<50aEUR%0nm distance between ER and mitochondria were considered as MAM). Mito: Mitochondria (red), ER: Endoplasmic Reticulum (green), Nucleus (blue). caEUR"e Quantification of average mitochondria perimeter (c) and ER perimeter (d), and percentage of mitochondria apposition to ER (e), naEUR%0=aEUR%015 microscopic fields each (number of mitochondria/ER analyzed, CD: 260/620 and ED: 390/1040 from 3 mice/group). f Magnified TEM images (Scale bars, 1aEUR%0Aum). M: Mitochondria, ER: Endoplasmic Reticulum. g Quantification of MAM length to mitochondrial perimeter ratio, naEUR%0=aEUR%015 microscopic fields each (number of mitochondria/ER analyzed, CD: 260/620 and ED: 390/1040 from 3 mice/group). h Confocal microscopy analysis of ER and mitochondria using PDI (Green) and TOM20 (Red) antibodies, respectively, in primary mouse hepatocytes cultured with or without 100aEUR%0mM EtOH for 24aEUR%0h (Scale bars, 20aEUR%0Aum). Colocalized portion of ER and M - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Enhanced Ca… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36973273), licensed under a CC BY license.


PDK4 deficiency suppresses EtOH-induced MAM formation and mitochondrial dysfunction.a Analysis of interaction between MCC complex proteins in Pdk4+/+ and Pdk4a^'/a^' primary mouse hepatocytes treated with or without 100aEUR%0mM EtOH for 24aEUR%0h by in situ PLA (Scale bars, 20aEUR%0Aum). baEUR"d Quantification of (a) [IP3R1-GRP75 (b), naEUR%0=aEUR%014; VDAC1-GRP75 (c), naEUR%0=aEUR%014; IP3R1-VDAC1 (d), naEUR%0=aEUR%015 microscopic fields with >300 cells from 3 independent experiments]. e Immunofluorescence analysis of ER and mitochondria using PDI (Green) and TOM20 (Red) antibodies in Pdk4+/+ and Pdk4-/- primary mouse hepatocytes treated with or without EtOH for 24aEUR%0h. Colocalized portion of ER and Mitochondria is shown in white (Scale bars, 20aEUR%0Aum). f Quantification of TOM20-PDI colocalization of (e) (Ctrl Pdk4+/+, naEUR%0=aEUR%028; Ctrl Pdk4a^'/a^', naEUR%0=aEUR%024; EtOH Pdk4+/+ and EtOH Pdk4a^'/a^', naEUR%0=aEUR%030 microscopic fields with >300 cells from 3 independent experiments). g Measurement of mitochondrial Ca2+ level in Pdk4+/+ and Pdk4a^'/a^' primary mouse hepatocytes transduced with 4mitD3 adenovirus and treated with or without EtOH for 24aEUR%0h (naEUR%0=aEUR%08 biological replicates from three independent experiments). h, i Mitochondria ROS (mean fluorescence intensity; MFI) (naEUR%0=aEUR%06 bio - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Enhanced Ca… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36973273), licensed under a CC BY license.


Alcohol augments hepatic ER-mitochondria interaction.a Graphical depiction of alcohol feeding model in mice. b Mitochondria and ER morphology in the perivenous region of control diet (CD) and EtOH diet (ED)-fed mice liver sections were visualized using TEM (Scale bars, 5aEUR%0Aum). The ER and mitochondria in the TEM images were reconstructed graphically to visualize MAM formation. (<50aEUR%0nm distance between ER and mitochondria were considered as MAM). Mito: Mitochondria (red), ER: Endoplasmic Reticulum (green), Nucleus (blue). caEUR"e Quantification of average mitochondria perimeter (c) and ER perimeter (d), and percentage of mitochondria apposition to ER (e), naEUR%0=aEUR%015 microscopic fields each (number of mitochondria/ER analyzed, CD: 260/620 and ED: 390/1040 from 3 mice/group). f Magnified TEM images (Scale bars, 1aEUR%0Aum). M: Mitochondria, ER: Endoplasmic Reticulum. g Quantification of MAM length to mitochondrial perimeter ratio, naEUR%0=aEUR%015 microscopic fields each (number of mitochondria/ER analyzed, CD: 260/620 and ED: 390/1040 from 3 mice/group). h Confocal microscopy analysis of ER and mitochondria using PDI (Green) and TOM20 (Red) antibodies, respectively, in primary mouse hepatocytes cultured with or without 100aEUR%0mM EtOH for 24aEUR%0h (Scale bars, 20aEUR%0Aum). Colocalized portion of ER and M - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Enhanced Ca… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36973273), licensed under a CC BY license.


Immunocytochemistry for GABA (A), GABAB receptors (B) and NMDA receptors (C) along with MAP2 neuronal cytoskeletal marker at 14 DIV. Scale bar = 200 µm. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Selective inhibition of excitatory synaptic transmission alters the emergent bursting dynamics of in vitro neural networks. Front Neural Circuits (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36874945), licensed under a CC BY license.


Adherent junction drives osmolarity-driven barrier function change. A) Number of genes that are upregulated by more than twofolds in hyper- (Hyper/Hypo > 2) and iso- (Iso/Hypo > 2) compared to hypo-osmotic conditions. B) Gene ontology (GO) analysis of the 862 intersected genes. Significantly enriched gene sets were selected from the PANTHER pathway. The dashed vertical lines indicate significance at p < 0.05. C) Gene set enrichment analysis (GSEA) results showing significant enrichment of the gene sets, “Cadherin signaling pathway” in Reactome from the Molecular Signatures Database (MSigDB) in iso- compared to hypo-osmotic conditions. Red and blue shading indicate high and low log2-ranked values comparing iso- to hypo-osmotic conditions, respectively. ES: enrichment score, NES: normalized enrichment score, and Nom p-value: nominal p-value. D) Heatmap visualization of gene expression profiles of PANTHER_Cadherin signaling pathway (P00012). Genes over 1.2-fold up- (Hyper/Hypo > 1.2) and downregulated (Hypo/Hyper > 1.2) in hyper- compared to hypo-osmotic conditions are displayed based on the z-score. E,F) Representative immunostaining of VE-cadherin in HUVEC 3D engineered microvessels and 2.5D monolayer (see the Experimental Section) 2 d after corresponding osmolarity adjustment (hypo-, iso-, and hyperosmolarity at D2; see Fi - Image collected and cropped by CiteAb under a CC-BY license from the following publicati… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36808839), licensed under a CC BY license.


Cell-cell junction localization of F-actin and actomyosin-dependent barrier function imply mechanobiological adaptation of microvessels during osmolarity exposure. A) Representative immunostaining of F-actin and VE-Cadherin in HUVEC 2.5D monolayer 2 d after corresponding osmotic adjustment (hypo-, iso-, or hyperosmotic condition at D2; see Figure 1B for detailed timelines). Scale bars, 50 µm. Cell nuclei were counterstained with DAPI. Inset: zoom-in view of F-actin and VE-cadherin (yellow) and actin and nucleus (cyan) colocalized pixels. Cell nuclei were counterstained with DAPI. Scale bars, 10 µm. B,C) Fraction of F-Actin & VE-cadherin and F-Actin & Nucleus colocalized pixels from the immunostained images. Mean ± S.D. n = 8 images from five biological replicates. See Figure S15 (Supporting Information) for detailed processing steps. D-F) Representative fluorescent images of 4 kDa FITC-dextran leakage from osmolarity-adapted HUVEC 3D engineered microvessels without treatment, 30 min after 10 × 10−6 m Blebbistatin, and 10 × 10−6 m Y-27632 treatment. t = 0 min images were taken immediately after the lumen was filled with FITC-dextran solutions. G,H) Barrier function changes, relative to iso-osmotic controls or before drug treatments, in osmolarity-adapted HUVEC 3D engineered microvessels 30 min - Image collected and cropped by CiteAb under a CC-BY license from the following publicati… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36808839), licensed under a CC BY license.


Hyperosmolarity-adapted microvessels display significantly improved barrier protection under acute and chronic inflammation. A) Experimental timeline for testing the barrier protective effect of osmolarity-adapted HUVEC 3D engineered microvessels following tumor necrosis factor alpha (TNFα) or lipopolysaccharides (LPS) induced vascular inflammations. Note that osmolarity was persistently maintained during inflammation. B) Western blot displaying ICAM-1 (an inflammatory marker), and VE-cadherin levels of osmolarity-adapted (hypo-, iso-, or hyperosmotic) 2.5D HUVEC monolayers 24 h after 0 or 5 ng mL−1 TNFα treatment. GAPDH was used as a loading control. C-E) Representative immunostaining of VE-cadherin and F-actin in osmolarity-adapted HUVEC 3D engineered microvessels 24 h after 5 ng mL−1 TNFα, 24 h after 100 ng mL−1 LPS treatment, and HUVEC 2.5D monolayer 24 h after 5 ng mL−1 TNFα treatment. Cell nuclei were counterstained with DAPI. Scale bars, 50 µm. F) Representative fluorescent images of 4 kDa FITC-dextran leakage from osmolarity adjusted HUVEC 3D engineered microvessels before (left) and 24 h after 5 ng mL−1 TNFα (right) treatment. t = 0 min images were taken immediately after the lumen was filled with 4 kDa FITC dextran. G) Barrier function before and 24 h after 5 - Image collected and cropped by CiteAb under a CC-BY license from the following publicati… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36808839), licensed under a CC BY license.


TECPR2 deficiency impacts the composition of lysosomes.a TECPR2 WT and MUT cells expressing C-terminally 3×HA or 2×FLAG-tagged TMEM192 were lysed and immunoblotted. Tubulin served as loading control. b TECPR2 WT and MUT cells expressing C-terminally 3×HA-tagged TMEM192 were fixed and immunolabeled. LAMP1 and LAMP2 served as lysosomal marker. Scale bar 10 µm. c Quantification of colocalization of LAMP1 and LAMP2 with 3×HA-tagged TMEM192 between TECPR2 WT and MUT cells per statistical two-sided t-test analysis of normalized overlap measures (n = 4 independent experiments). Error bars represent mean ± SEM, p values = 0.75034 and 0.77539 (for LAMP1) and 0.91922 and 0.91032 (LAMP2). d Scheme of LysoIP workflow. e Venn graph-like summary of proteins enriched by TMEM192-3×HA over TMEM192-2×FLAG background binding (gray circle) and of proteins increased (red circle) and decreased (blue circle) in TECPR2 WT and MUT cells, respectively. f Volcano plot of proteins identified by LysoIP in TECPR2 WT and MUT cells. Significantly altered proteins (two-sided t-test, n = 4 independent experiments) are grouped as class I (dark color) or class II (light color). Known lysosomal components and selected candidates are highlighted. g Total abundance of altered LysoIP candidates (in percent) in the whole cell proteomic analysis of TECP - Image collected and cropped by CiteAb under a CC-BY license fr… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36797266), licensed under a CC BY license.


High molecular weight p21L intron-retained isoform with an alternative C-terminus loses its nuclear localisation and consequently its CDK inhibitory activity. (A) Growth curves of Nalm-6 parental and Nalm-6-p21L (transduced) cells with 0.5 µg/mL or without doxycycline (-dox) in the medium. The number of viable cells was assessed by trypan blue exclusion. Error bars show the mean ± standard error of the mean (SEM) of three independent counts of viable cells observed under trypan blue exclusion assay. (B) Western blots of parental Nalm-6 and Nalm-6-p21L (transduced) either noninduced or induced with 0.5 µg/mL doxycycline for 24 h, showing the expression of the intron-retained variant of p21 in the Nalm-6-p21L transduced cells. MDM2 inhibitor RG7388 (0.3 µM) was used to induce endogenous wild-type p21WAF1. RB phosphorylation was suppressed by the induced endogenous p21WAF1, but not by the transduced p21L isoform. Actin was used as a loading control. (C) The aberrant p21L isoform was unable to localize to the nucleus. RG7388 was used to induce endogenous p21, which showed nuclear localisation. Twenty-four hours after doxycycline and RG7388 administration, cells were fixed on | Image collected and cropped by CiteAb under a CC-BY license from the following publication: Splicing Modulation Results in Aberrant Isoforms and Protein Products of p53 Pathway Genes and the Sensitization of B Cells to Non-Genotoxic MDM2 Inhibition. Int J Mol … Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Expression of cell specific markers in single cultures of bull PB-MSC, SSC with SC. (A) Gene expression levels of SC markers WT1 and RA were higher (p < 0.05) in SC and WT1 and were not detected in SSC or MSC. Gene expression of SSC gene marker UCHL1 was higher (p < 0.05) in SSC. CD90 gene expression was only expressed in SSC. Levels of MSC CD73 gene marker were similar (p > 0.05) between SC and MSC and were not detected in SSC. CD105 gene marker had higher expression (p < 0.05) in MSC compared to SC. CD45 gene marker had higher expression (p < 0.05) in SC comparing with SSC and MSC, whereas CD34 gene had higher expression (p < 0.05) in SSC compared to SC and MSC. (B) Immunofluorescence associated to UCHL1 was observed in SSC. (C) Intense, Wt1 immunofluorescence was observed in SC. (D) Immunofluorescence associated to CD73 was observed in MSC. (*) Indicate gene expression was not detected for a single marker. Different superscripts (a, b, c) indicate differences (p < 0.05) for the same marker between cell types. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC). Animals (Basel) (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36670859), licensed under a CC BY license.


Expression of cell specific markers in single cultures of bull PB-MSC, SSC with SC. (A) Gene expression levels of SC markers WT1 and RA were higher (p < 0.05) in SC and WT1 and were not detected in SSC or MSC. Gene expression of SSC gene marker UCHL1 was higher (p < 0.05) in SSC. CD90 gene expression was only expressed in SSC. Levels of MSC CD73 gene marker were similar (p > 0.05) between SC and MSC and were not detected in SSC. CD105 gene marker had higher expression (p < 0.05) in MSC compared to SC. CD45 gene marker had higher expression (p < 0.05) in SC comparing with SSC and MSC, whereas CD34 gene had higher expression (p < 0.05) in SSC compared to SC and MSC. (B) Immunofluorescence associated to UCHL1 was observed in SSC. (C) Intense, Wt1 immunofluorescence was observed in SC. (D) Immunofluorescence associated to CD73 was observed in MSC. (*) Indicate gene expression was not detected for a single marker. Different superscripts (a, b, c) indicate differences (p < 0.05) for the same marker between cell types. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC). Animals (Basel) (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36670859), licensed under a CC BY license.


Expression of cell specific markers in single cultures of bull PB-MSC, SSC with SC. (A) Gene expression levels of SC markers WT1 and RA were higher (p < 0.05) in SC and WT1 and were not detected in SSC or MSC. Gene expression of SSC gene marker UCHL1 was higher (p < 0.05) in SSC. CD90 gene expression was only expressed in SSC. Levels of MSC CD73 gene marker were similar (p > 0.05) between SC and MSC and were not detected in SSC. CD105 gene marker had higher expression (p < 0.05) in MSC compared to SC. CD45 gene marker had higher expression (p < 0.05) in SC comparing with SSC and MSC, whereas CD34 gene had higher expression (p < 0.05) in SSC compared to SC and MSC. (B) Immunofluorescence associated to UCHL1 was observed in SSC. (C) Intense, Wt1 immunofluorescence was observed in SC. (D) Immunofluorescence associated to CD73 was observed in MSC. (*) Indicate gene expression was not detected for a single marker. Different superscripts (a, b, c) indicate differences (p < 0.05) for the same marker between cell types. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC). Animals (Basel) (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36670859), licensed under a CC BY license.


Influence of PNGase F digestion and tunicamycin treatment on the glycosylation status and localization of ABCB1 protein in DU145 and PC-3 DTXR cells. (a) Protein lysates of DU145 and PC-3 DTXR cells were treated with PNGase F for 2 h and compared to untreated protein lysates (UT). An exemplary Western blot for the detection of ABCB1 protein with GAPDH as reference protein is shown. (b) DU145 and PC-3 DTXR cells were pre-treated with 100 ng/mL tunicamycin (TUN) for 96 h and compared to untreated cells (UT). An exemplary Western blot for detecting ABCB1 protein with I+--tubulin as reference protein is shown. (a,b) The protein bands were analyzed densitometrically and then normalized to the respective reference protein. The different glycosylation statuses are depicted as part of the total ABCB1 amount in %. (c) Immunofluorescence staining of ABCB1 in DU145 and PC-3 CTRL cells as well as DTXR cells with or without pre-treatment with 100 ng/mL tunicamycin (TUN) for 96 h. Cell nuclei were visualized with DAPI (blue), ABCB1 was detected using an anti-ABCB1 antibody and an Alexa Fluor 488-conjugated secondary antibody (green) and then the images were merged (scale bar 40 Aum). Exemplary images and data, which are depicted as mean A+- SD, are taken from four to five independent biological replicates. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Comprehensive Evaluation of Multiple Ap… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36614114), licensed under a CC BY license.


Motor proteins in TNTs of normal (NPU) and cancer (T24) urothelial cells. NPU and T24 cells contain TNTs with myosin Va (A), dynein (B), and kinesin 5B (C) along with F-actin or α-tubulin. The colocalization between motor proteins and cytoskeletal elements in TNTs (yellow arrowheads) from the insets in the merged images are magnified in the corners. The nuclei were stained with DAPI. Scale bars: 20 µm (D) Quantification of fluorescent dots of motor proteins (arbitrary units) per µm of TNT (mean ± SEM; NPU: kinesin, n = 17; dynein, n = 19; myosin, n = 16; T24: kinesin, n = 10; dynein, n = 13; myosin, n = 15; *p < 0.05, **p < 0.01). - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Molecular, morphological and functional properties of tunnelling nanotubes between normal and cancer urothelial cells: New insights from the in vitro model mimicking the situation after surgical removal of the urothelial tumor. Front Cell Dev Biol (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36601539), licensed under a CC BY license.


Mitochondria, lysosomes and Golgi apparatus in TNTs of normal (NPU) urothelial cells. Distribution of mitochondria (A,D,D’), lysosomes (B), and Golgi apparatus (C) in TNTs. The large insets in A, B, C are 300% magnifications of the corresponding small insets. (D) The distribution of mitochondria and microtubules in TNTs, enlarged in (D’). Numbers 1, 2, and 3 (arrows) denote cross sections in TNT where profiles of fluorescence intensities of microtubules and MitoTracker were measured (E). Data is presented as intensity of fluorescence (arbitrary units a. u.) per 1.8 µm cross section. Scale bars: 20 µm. - Image collected and cropped by CiteAb under a CC-BY license from the following publication: Molecular, morphological and functional properties of tunnelling nanotubes between normal and cancer urothelial cells: New insights from the in vitro model mimicking the situation after surgical removal of the urothelial tumor. Front Cell Dev Biol (2023) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36601539), licensed under a CC BY license.


Qualitative and quantitative analyses of nuclear rim colocalization patterns: coexpressed sequence-predicted hybrid constructs (HCMV/EBV pUL53 sHook1 and HCMV/VZV pUL53 sHook2) in combination with three different groove proteins (HCMV pUL50, EBV BFRF1 or VZV Orf24). (A) HeLa or (B) 293T cells were transiently cotransfected with either constructs coding for the Flag-tagged sequence-predicted hooks, sHook1 or sHook2, and the HA-tagged groove proteins pUL50, BFRF1 or Orf24. Two d p.t., cells were fixed, used for immunostaining with tag-specific antibodies and analyzed by confocal imaging. DAPI counterstaining indicated the morphology of nuclei of the respective cells. In both types of cells, the localization patterns of coexpressed construct pairs were analyzed; the scale bar in panels A-C, picture 1 marks 7.5 µm. On a qualitative level of analysis (A,B), the yellow arrows indicate complete nuclear rim colocalization, and the white arrows show partial rim colocalization. (C) Staining of subcellular localizations of marker proteins in HeLa and 293T cells, i.e., the nuclear lamina, the nucleoplasmic and cytosolic compartments. (D) Quantitation of the colocalization patterns of pUL53 sHook1 and sHook2 with groove proteins HCMV pUL50, EBV BFRF1 and VZV Orf24. Localization patterns observed in HeLa and 293T cells were quantified and c - Image collected and cropped by CiteAb under a CC-BY license from the following publication… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36552794), licensed under a CC BY license.


Dhcr7 microglia exhibit immune activation and markers of reactivity. Purified microglia from Dhcr7 mutant mice display hallmark signs of activation. (A) CellMask (blue) staining and CD68 (red) immunolabeling of control and Dhcr7 microglia. Scale bar: 100 µm. (B,C) High-content analyses showed cellular hypertrophy and increased CD68 expression/cell in Dhcr7 microglia following culture in LPDS conditions (one-way ANOVA and Dunnett's test versus LPDS control; n=12 images for cell area and n=6 images for CD68 intensity from three biological replicates). (D) qRT-PCR analysis shows increased expression of reactive transcripts in Dhcr7 mutant microglia samples (one-way ANOVA and Dunnett's test versus LPDS control; n=3 biological replicates; each data point represents the average of three technical replicates). (E) CD68 and Iba1 immunolabeling of P7 and P30 brain sections (cortex) of control and Dhcr7 mice. Scale bar: 25 µm. (F,G) Analysis of CD68 and Iba1 intensity normalized to the area of the image field shows increased intensity in Dhcr7 mutant brains at both timepoints (two-tailed unpaired t-test; n=15 images from three biological replicates). Data show the mean±s.e.m. a.u., arbitrary units. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


WWP2 regulates macrophage activation and profibrotic function.a Scatter plot of log2fold changes (FC) in mRNA expression from scRNA-seq in cardiac macrophages between Ang-II-treated Mut/Mut and WT mice (y axis) and log2FC between WT Ang-II-treated and WT untreated mice (x axis). Differentially expressed genes (DEGs) in red (n = 237, FDR < 0.05). Ang-II treatment: 500 ng/kg/min, 7 days. b Top downregulated pathways in Mut/Mut macrophages identified by gene set enrichment analysis (GSEA) of DEGs. NES, normalized enrichment score. c Violin plots illustrate the expression score of the GSEA-derived pathways across all cardiac macrophage clusters in Mut/Mut and WT mice after treatment with Ang-II (7 days). d qRT-PCR analysis of selected pro-inflammatory and homeostatic/reparatory genes in macrophages sorted from LV of WT and Mut/Mut mice treated with saline or Ang-II (7 days). n = 5-12 for each group. e Representative immunofluorescence staining of smooth muscle aortic alpha-actin (ACTA2, green) in (myo)fibroblasts co-cultured with CD45 + macrophages (red). Scale bar, 100 µm. f Number of cardiac macrophages moving across the proximity border (left), and ACTA2 expression in (myo)fibroblasts (right). n = 3 per experimental group and 15-25 fibroblast images were taken from each slide. g Schematic of the co-culture experimental setup in vitro. The conditioned supernatant (CS) from BMDMs treated with LPS (100 ng/ml, 4 hrs) and I… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36450710), licensed under a CC BY license.


WWP2 regulates macrophage activation and profibrotic function.a Scatter plot of log2fold changes (FC) in mRNA expression from scRNA-seq in cardiac macrophages between Ang-II-treated Mut/Mut and WT mice (y axis) and log2FC between WT Ang-II-treated and WT untreated mice (x axis). Differentially expressed genes (DEGs) in red (n = 237, FDR < 0.05). Ang-II treatment: 500 ng/kg/min, 7 days. b Top downregulated pathways in Mut/Mut macrophages identified by gene set enrichment analysis (GSEA) of DEGs. NES, normalized enrichment score. c Violin plots illustrate the expression score of the GSEA-derived pathways across all cardiac macrophage clusters in Mut/Mut and WT mice after treatment with Ang-II (7 days). d qRT-PCR analysis of selected pro-inflammatory and homeostatic/reparatory genes in macrophages sorted from LV of WT and Mut/Mut mice treated with saline or Ang-II (7 days). n = 5-12 for each group. e Representative immunofluorescence staining of smooth muscle aortic alpha-actin (ACTA2, green) in (myo)fibroblasts co-cultured with CD45 + macrophages (red). Scale bar, 100 µm. f Number of cardiac macrophages moving across the proximity border (left), and ACTA2 expression in (myo)fibroblasts (right). n = 3 per experimental group and 15-25 fibroblast images were taken from each slide. g Schematic of the co-culture experimental setup in vitro. The conditioned supernatant (CS) from BMDMs treated with LPS (100 ng/ml, 4 hrs) and I… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36450710), licensed under a CC BY license.


Morphological analysis of young RTT neurons. (a) IF of MAP2 (red), TUJ1 (green), and DAPI (blue). Scale bar: 50 µm. (b-d) Box-plots from the comparison between mutant (red) versus isogenic WT clones (light grey) of RTT females and between the male (blue) versus controls (dark grey) for end points number, branches number and neuronal length. Decreased number of end points (b), branches (c), and neuronal length (d) were highly significant in the Kruskal-Wallis test, while no significance was proved in the comparison among wild-type clones. Data were compared using Kruskal-Wallis test. In (b) **** p-value < 0.0001, *** p-value = 0.0005; in (c) **** p-value < 0.0001, *** p-value = 0.0003; in (d) **** p-value < 0.0001, * p-value = 0.0475 and 0.0161. ns: not significant. A correction was also introduced by Bonferroni test. Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Nuclear area size of young RTT neurons. (a) IF staining with pan-neuronal markers MAP2 (red) and TUJ1 (green), counterstaining with DAPI and analysis with Image J software of single young neurons (top panels) and scanned nuclear area size (bottom panels) of a control, PtY2 (p.Gly252Argfs*7), PtA2 Mut and PtB1 Mut i-Neurons. Scale bar: 50 µm. (b) Box-plots representative of the comparison for nuclear area of each mutant clone and a pool of eight healthy controls, four males and four females (CTRLs), (c) each isogenic WT clone vs. the correspondent mutant carrying the p.Arg255* and p.Arg133Cys, (d) between each isogenic WT clone and a pool of controls, (e) across WT isogenic clones. Data were compared using Kruskal-Wallis test. **** p-value < 0.0001, *** p-value = 0.0004, ** p-value = 0.0058. ns: not significant. Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


MeCP2 protein in i-neurons from RTT cases with truncating and missense variants. (a,c) Western blot on protein extracted from neurons of controls (CTRL), patients carriers of p.Gly252Argfs*7 (PtY), p.Arg255* (PtA1), p.Arg133Cys (PtB1), wild type (WT) and mutant (Mut) isogenic clones, using N-ter antibody (Ab) (a) and C-ter Ab (c). The blue rectangles frame the band representative of the full-length protein (~75 kDa), while the red ones frame the 37 kDa truncated isoform. Histone H3 (15 kDa) was used as loading control. Panels (b) and (d) show immunostaining of mature neurons with beta 3 tubulin (TUJ1), highlighting the localization of MeCP2 (red). Nuclei were counterstained with DAPI; scale bar: 20 µm. Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


MeCP2 protein in i-neurons from RTT cases with truncating and missense variants. (a,c) Western blot on protein extracted from neurons of controls (CTRL), patients carriers of p.Gly252Argfs*7 (PtY), p.Arg255* (PtA1), p.Arg133Cys (PtB1), wild type (WT) and mutant (Mut) isogenic clones, using N-ter antibody (Ab) (a) and C-ter Ab (c). The blue rectangles frame the band representative of the full-length protein (~75 kDa), while the red ones frame the 37 kDa truncated isoform. Histone H3 (15 kDa) was used as loading control. Panels (b) and (d) show immunostaining of mature neurons with beta 3 tubulin (TUJ1), highlighting the localization of MeCP2 (red). Nuclei were counterstained with DAPI; scale bar: 20 µm. Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Effects of paclitaxel on astrocytes. (A) Relative expression level of Connexin 43 (CX43) in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 6). (B) Released adenosine triphosphate (ATP) in µM after paclitaxel exposure with 1 µM and 5 µM compared to untreated (CTRL) cells (n (number of biological replicates) = 17). (C) Relative expression level of glial fibrillary acidic protein (GFAP) in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 5). (D) Relative expression level of vimentin in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 5). (E) Relative expression level of Complement 3 (C3) in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control. (F) Relative expression level of S100A10 in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 4). (G) Immunofluorescence micrographs of CX43 and GFAP in astrocyte cultures after no exposure (CTRL) or exposure to 1 µM and 5 µM paclitaxel (PCX). (H) Immunofluorescence micrographs of C3 and GFAP in astrocyte cultures after no exposure (CTRL) or exposure to 1 µM and 5 µM paclitaxel (PCX). (I) Immunofluorescenc… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Effects of paclitaxel on satellite glial cells. (A) Immunofluorescence micrographs of Connexin 43 (CX43) and glial fibrillary acidic protein (GFAP) in satellite glial cells (SGC) after no treatment or exposure to 1 µM and 5 µM paclitaxel (PCX). (B) Relative expression level of GFAP in SGC after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 3). (C) Relative expression level of CX43 in SGC after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 3). (D) Released adenosine triphosphate (ATP) in µM after paclitaxel exposure to 1 µM and 5 µM compared to untreated (CTRL) cells (n (number of biological replicates) = 12). (E) Heatmap of pro-inflammatory markers, tumor necrosis factor-α (TNF-α), Interleukin-1 β (IL-1β), IL-6 and iNOS, and anti-inflammatory, IL-10, expression after 1 µM and 5 µM paclitaxel exposure depicted as 2(−ΔΔCT). Measured relative amount of pro-inflammatory TNF-α (F) (n (number of independent cell culture preparations) = 3), IL-1β (G) (n (number of independent cell culture preparations) = 6), and IL-6 (H) (n (number of independent cell culture preparations) = 4) via enzyme-linked immunosorbent assay (ELISA). For an easier interpretation of the data, pro-inflammatory markers are depicted in red, anti-inflammatory mar… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Effects of paclitaxel on microglia. (A) Relative expression level of Connexin (CX43) in microglia after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 6). (B) Released adenosine triphosphate (ATP) in µM after paclitaxel exposure with 1 µM and 5 µM compared to unexposed (CTRL) cells (n (number of biological replicates) = 10). (C) Immunofluorescence micrographs of CX43 and vimentin in microglia after no exposure (CTRL) or exposure to 1 µM and 5 µM paclitaxel (PCX). (D) Immunofluorescence micrographs of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB1) and ionized calcium-binding adapter molecule 1 (Iba1) in microglia after no exposure or exposure to 1 µM and 5 µM PCX. (E) Immunofluorescence micrographs of iNOS and Iba1 in microglia after no exposure or exposure to 1 µM and 5 µM PCX. (F) Heatmap of pro-inflammatory markers, tumor necrosis factor-α (TNF-α), Interleukin-1 β (IL-1β), IL-6 and iNOS, and anti-inflammatory, IL-10 and cluster of differentiation 206 (CD206), expression after 1 µM and 5 µM paclitaxel treatment depicted as 2(−ΔΔCT). For an easier interpretation of the data, pro-inflammatory markers are depicted in red, anti-inflammatory markers are depicted in green. Measured relative amount of pro-inflammatory TNF-α (G), IL-1β (H), and IL-6 (I)… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Effects of paclitaxel on astrocytes. (A) Relative expression level of Connexin 43 (CX43) in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 6). (B) Released adenosine triphosphate (ATP) in µM after paclitaxel exposure with 1 µM and 5 µM compared to untreated (CTRL) cells (n (number of biological replicates) = 17). (C) Relative expression level of glial fibrillary acidic protein (GFAP) in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 5). (D) Relative expression level of vimentin in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 5). (E) Relative expression level of Complement 3 (C3) in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control. (F) Relative expression level of S100A10 in astrocytes after 1 µM and 5 µM paclitaxel exposure compared to control (n (number of independent cell culture preparations) = 4). (G) Immunofluorescence micrographs of CX43 and GFAP in astrocyte cultures after no exposure (CTRL) or exposure to 1 µM and 5 µM paclitaxel (PCX). (H) Immunofluorescence micrographs of C3 and GFAP in astrocyte cultures after no exposure (CTRL) or exposure to 1 µM and 5 µM paclitaxel (PCX). (I) Immunofluorescenc… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


IRAP-deficient BMMCs show diminished phosphorylation of Syk at the plasma membrane where it colocalizes with IgE. (A) WT and IRAP-deficient (IRAPKO) BMMCs were sensitized with anti-DNP IgE and challenged with antigen (30 ng/mL of DNP-HSA). Stimulation was arrested at indicated time points and cellular lysates were prepared in SDS sample buffer. Western Blot analysis (left panel) show pSyk staining (anti-pSykY519/520) and total Syk determined after stripping. Quantitative data (right panel) are the ratio between pSyk/Syk and were normalized by setting non stimulated values to 1. They represent the mean ± s.e.m of indicated experiments. Statistical analysis was done using the Wilcoxon test. (B) Anti-DNP IgE sensitized WT (blue) and IRAPKO BMMCs (red) were loaded with indo-1 and stimulated after addition (arrow) of 30 ng/mL of DNP-HSA. The fluorescence emission ratio for Ca2+-bound/Ca2+-free indo-1 was measured. Similar results were obtained in three independent experiments. (C) Anti-DNP IgE sensitized WT and IRAPKO BMMCs were plated on fibronectin-coated glass coverslips. Cells were stimulated with 30 ng/mL of DNP-HSA for indicated time points. After fixation and permeabilization cells were stained with DAPI (blue), anti-IgE (green) and anti-pSykY519/520 (red) as indicated. Cells were analyzed by confocal microscopy. Images show representative sections with multiple cells for IgE and pSykY519/520 single staining as well … Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36389775), licensed under a CC BY license.


Y136DOPA modification of overexpressed mCh-αSyn detected by a specific antibody.a A specific antibody for Y136DOPA of αSyn. Y136DOPAmab specifically recognized TH-treated (TH +) but not control (TH-) αSyn, E46K, and A53T in WB analysis. b, c Y136DOPA signals detected in cultured SCG neurons (b) and PC12 cells (c). SCG and PC12 cells overexpressing mCh-αSyn by the lentivirus system were harvested at Day (d) 4 or 8 after infection and continuously examined by WB. The Y136DOPA signal was slightly detected at d4 but significantly increased at d8. βIII-tubulin is a loading control. d Immunofluorescence of PC12 cells overexpressing mCherry (control, upper) or mCh-αSyn (lower) by the lentivirus system. The Y136DOPA signal (green) was detected in cells expressing mCh-αSyn (red) at d8 after infection. Scale bar: 20 µm. e Dopanization of mCh-αSyn in mouse SN neurons. mCh-αSyn was strongly overexpressed in the SNc by the stereotaxic injection of AAV. WB using the SN tissue detected Y136DOPA at 5 weeks after injection. f Immunohistochemistry staining of AAV-injected brain sections. Y136DOPA modification (green) of mCh-αSyn (red) was detected in the cell bodies of TH-positive dopaminergic neurons (blue) in the SNc (upper, white arrowheads), as well as their nerve terminals (white) in the striatum (lower, blue arrows). Scale bars: 20 µm (upper) and 10 µm (lower). g Y136DOPA signals decreased after tre… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


EGCG inhibits autophagy and mitophagy-related molecules.A qRT-PCR for various gene expressions is presented graphically. B Representative IF staining images is presented. C Quantified mean intensity graphs for IF staining is presented graphically. D WB images are shown for protein levels of ATG5, ATG7, Beclin1, LC3B, PINK1, PARKIN, DRP1, and FIS1 during the OC differentiation in the presence or absence of EGCG. For qRT-PCR, β-actin, and WB, GAPDH was used as a loading control. Results are shown in mean ± SEM (n = 3). (#), p < 0.05 compared with basal condition; and (*), p < 0.05 compared with induced OC differentiated condition. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36307395), licensed under a CC BY license.


Tethering RUVBL2 to an active gene promoter enhances Pol II cluster formation.a Schematic showing the system used for simultaneous imaging of tethered proteins and recruited proteins, as published previously60. b Representative images of endogenous RUVBL1 and RUVBL2. Scale bar, 5 µm. c The relative IF signals of the target proteins at the LacO loci under mock (RUVBL1 n = 24, RUVBL2 n = 30) and Dox treatments (RUVBL1 n = 18, RUVBL2 n = 30). Box plots indicate median (middle line), 25th and 75th percentile (box) and 5th and 95th percentile (whiskers), as well as outliers (single black points), differences between groups were analyzed by two-tailed Student’s t tests (****p < 0.0001). d The mean and total intensity of LacR-BFP fusion signals at LacO loci per cell were plotted in bar graphs. The cotransfected cells (n = 30) in different fields from 3 transfection replicates were used for quantification. Differences between groups were analyzed by two-tailed Student’s t tests. “ns” indicates no significant difference. The error bars indicate mean + /− SDs. e Representative images of endogenous Pol II before and after transcriptional activation. Scale bars, 5 µm. f The relative IF signals of Pol II and LacR-BFP fusions at the LacO loci were plotted as boxplot. Cells from a minimum of 10 different fields (BFP n = 11, RUVBL1 n = 28, RUVBL2 n = 29 in mock state, and BFP n = 13, RUVBL1 n = 24,… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36171202), licensed under a CC BY license.


Identification of direct target genes of the RUVBL2-Pol II axis.a Workflow of the process used to identify the genes directly targeted by RUVBL2. The FDR are extracted from DESeq2 by adjusting p-values using BH method. b Heatmap analyses of RUVBL2 direct target genes based on PRO-Seq expression. c Snapshots showing the RUVBL1 and RUVBL2 ChIP-Seq signals and nascent RNA (PRO-Seq) signals at the Bmp4 and c-Myc loci after RUVBL2 degradation. d ChIP-Seq signals in the promoter regions of 45 genes directly targeted by RUVBL2 and 45 nontargeted genes in 153 datasets obtained from the Cistrome DB (Supplementary Data 5). e The 10 factors with the largest and smallest coefficients in the elastic net were selected. The heatmap shows the relative occupancy of these 10 factors in the 45 genes directly targeted by RUVBL2 and in 45 nontarget genes. f Pie chart indicating the percentage of C-MYC or CXXC1 ChIP-Seq-determined target genes that overlap the RUVBL2-affected genes as determined by the RNA-Seq data shown in Fig. 6g. g Profiles of PRO-Seq signals at the gene regions of directly targeted genes (n = 45), all genes (n = 36821) and posttranscriptional early response genes (n = 41) after RUVBL2 degradation. The profile above 0 indicates the signal on the sense strand, the profile below 0 indicates the signal on the antisense strand. Two-tailed Wilcoxon tests was performed to calculate the significance (*p < 0.05, **p < 0.01, ***p < 0… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36171202), licensed under a CC BY license.


RUVBL2 depletion decreases the number of Pol II clusters and inhibits global transcription.a Representative confocal microscopy images of Pol II (left) and CTCF (right) after RUVBL2 degradation. Scale bar, 3 µm. b The number of clusters of Pol II (left) and CTCF (right) per cell was quantified and shown as boxplot, all the cells data were shown as single black points (Pol II n = 15, 14, 13, respectively; CTCF n = 12, 13, 16, respectively). Two-tailed Student’s t tests were performed to determine the significance. (ns, not significant, **p < 0.01, ***p < 0.001). c The nuclear densities of Pol II per cell were measured in untreated and auxin-treated cells. The data represent 3 biological replicates and cells from different fields (n = 45, 43, 29, respectively) were used for each quantification. Two-tailed Student’s t tests were performed to determine the significance of differences between groups, and the error bars indicate the mean + /− SDs (*p < 0.05, **p < 0.01). d Volcano plot illustrating the results of the differential gene expression analysis using PRO-Seq after RUVBL2 depletion. FDR < 0.05 and |Fold Change (FC)| > 1.5 are used to reported the differential genes. The FDR are extracted from DESeq2 by adjusting p-values using BH method. e The results of metagene analyses of the PRO-Seq signals. The profile above 0 indicates the signal on the sense strand, the profile below 0 indicates the signal … Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36171202), licensed under a CC BY license.


Loss of CAF-1 suppresses genomic instability in BRCA-deficient cells.a, b γH2AX immunofluorescence experiment showing that CHAF1A depletion suppresses CPT-induced DNA damage accumulation in BRCA1 or BRCA2-depleted cells. HeLa cells were treated with 1 µM CPT for 1 h followed by media removal and chase in fresh media for 3 h. Representative micrographs (scale bar represents 50 µm) (a) and quantifications (b) are shown. At least 50 cells were quantified for each condition. The mean values are represented on the graph, and the p-values (t-test, two-tailed, unpaired) are listed at the top. Western blots confirming the co-depletions are shown in Supplementary Fig. 2a. c-e Neutral comet assay showing that CHAF1A depletion suppresses CPT-induced (c, d) and cisplatin-induced (e) DSB formation in BRCA1 or BRCA2-depleted cells. HeLa cells were treated with 100 nM CPT for 4 h or 2.5 µM cisplatin for 24 h. Representative micrographs (c) and quantifications (d, e) are shown. At least 60 nuclei were quantified for each condition. The mean values are represented on the graph, and the p-values (t-test, two-tailed, unpaired) are listed at the top. f, g Clonogenic survival experiments showing that CHAF1A co-depletion in BRCA2-knockdown (f) or BRCA1-knockdown (g) HeLa cells promotes cisplatin resistance. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate sta… Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36085347), licensed under a CC BY license.


Liver morphology: total hepatocyte, binucleated hepatocyte, other cells, and total cell number malondialdehyde per mm2 of liver tissue (A), representative images of liver tissue stained with Goldner trichrome (B), liver Caspase3 activity (C), the expression (D) and percentage of immunoreactive cells to autophagy-related protein LC3B (E), the expression (F) and percentage of immunoreactive cells to Beclin1 protein (G) in weaned rats not prenatally exposed to ACR (0-ACR) or exposed to ACR from the 6th day (L-ACR), 11th day (M-ACR) or 16th day (S-ACR) of prenatal life up to parturition. (B, D, F) All scale bars represent 100 µm. (A, C, E, F) A p-value was attributed above plots when two groups exhibit significant differences (p-value < 0.05). Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Inhibiting ion-channel activity significantly reduces cell migration speed in high viscosity media. A) Representative immunofluorescence images of NKCC1, NHE1, NaK and F-Actin of cells in DMEM and 1% MC. B) Cell speed upon inhibition of NKCC1 via 30 µm Bumetanide (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM 30 µm Bum N = 171; 1% MC 30 µm Bum N = 263). C) Cell speed upon inhibition of NHE1 via 20 µm EIPA (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM 20 µM EIPA N = 293; 1% MC 20 µm EipA N = 346). D) Cell speed upon dual- inhibition of NKCC1 and NHE1 via 30 µm Bumetanide and 20 µm EIPA (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM Dual N = 264; 1% MC Dual N = 461). E) Cell speed upon inhibition of NaK via 25 µm and 50 µm Ouabain (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM 25 µm Ouabain N = 202; 1% MC 25 µm Ouabain N = 288; DMEM 50 µm Ouabain N = 248; 1% MC 50 µm Ouabain N = 331). F) Cell speed upon NKCC1 knockdown from shRNA (DMEM Scr Ctrl N = 74; 1% MC Scr Ctrl N = 224; DMEM S1 N = 74; 1% MC S1 N = 301; DMEM S2 N = 71; 1% MC S2 N = 217; DMEM S3 N = 77; 1% MC S3 N = 133). G) Cell speed for NKCC1 knockdown cells with 20 µm EIPA (DMEM Scr Ctrl N = 14; 1% MC Scr Ctrl N = 17; DMEM S1 N = 13; 1% MC S1 N = 166; DMEM S2 N = 117; 1% MC S2 N = 188; DMEM S3 N = 84; 1% MC S3 N = 16. Image collected and cropped by CiteAb under a CC-BY license from the… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Detection of CYP inhibition after azamulin (AZA) treatment.a Schematic diagram of a possible mechanism of AZA binding with CYP. Active CYPs maintain a ferric low-spin (LS) state by using water as a ligand. After AZA enters the enzymatic pocket, water is replaced by AZA, which causes LS to high-spin (HS) transition. Although metabolic intermediates of AZA were not identified, the crystal structure indicated the pleuromutilin head is required for mechanism-based inhibition. The possible ligation was depicted as a black dotted line. b The Raman shifts at 1370 cm−1 and 1636 cm−1 decreased after treatment with AZA. c Comparison of average Raman signal at 1636 cm−1 and CYP3A4 activity assay. Error bars indicate SD between triplicates. ***P < 0.001; **P < 0.01. d Reconstructed Raman images at 600 cm−1, 675 cm−1, 1370 cm−1, and 1636 cm−1. Scale bars, 20 µm. e Immunostaining of cyt b5 and CYP3A4 at the same positions of Raman measurement. Scale bars, 20 µm. Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Measurement of CYP induction by Raman microscopy.a Average Raman spectra of HepaRG cells treated with (red) or without (gray) rifampicin (RIF). The Raman shifts of 1370 cm−1 and 1636 cm−1 increased after treatment with RIF for 48 h; enlarged in the insets. b Reconstructed Raman images at 600 cm−1, 675 cm−1, 1000 cm−1, 1370 cm−1, and 1636 cm−1, which can be assigned to cyt c, cyt b5, phenylalanine, oxidized CYPs, and LS CYPs, respectively. The comparison of these cellular components demonstrated that 1370 cm−1 and 1636 cm−1 increased after CYP induction. Scale bars, 20 µm. c Immunofluorescence staining of cyt b5 and CYP3A4 at the same positions as Raman measurement. Scale bars, 20 µm. d Western blots of CYP3A4, cyt c, cyt b5, and β-action. e CYP3A4 activity assay indicates CYP induction after RIF treatment. Error bars indicate SD between triplicates. ***P < 0.001. Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Motif analysis for identifying new binding partners of USP7.A Schematic overview of the workflow for identifying new binding partners of USP7 using binding motifs, DAVID, protein-protein interaction, and binding analyses. B The diagram shows the number of proteins having each or both [P/A/E]-X-X-S and K-X-X-X-K motifs. C Pathway enrichment of putative substrate proteins of USP7 was analyzed by DAVID analysis. Y-axis indicates the pathway name, and X-axis indicates gene count in the pathways, and the asterisk indicates the p-value (*p < 0.05, **p < 0.01, ***p < 0.001.) D Interaction network between USP7 and putative substrates with high interaction score and with or without the connection revealed. The color of nodes indicates the confirmation of binding between USP7 and proteins (Gray: published and green: nonpublished), and Raf-1 binding with USP7 is shown in purple. Interaction network is shown by Cytoscape. E Endogenous USP7 or Raf-1 was precipitated with an anti-USP7 or an anti-Raf-1 antibody in HeLa cell lysate. F HEK293T cells were transfected with Myc-USP7, and Flag-Raf-1 and immunoprecipitations were performed by an anti-Myc or an anti-Flag antibody. G Purified GST- or GST-USP7 expressed in BL21 was incubated with Flag-Raf-1-overexpressed HEK293T cells. GST- or GST-USP7 was pull downed by GST bead and gel was stained by Coomassie Brilliant Blue. H Immunocytochemical analysis was performed to investigate the localization of… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Motif analysis for identifying new binding partners of USP7.A Schematic overview of the workflow for identifying new binding partners of USP7 using binding motifs, DAVID, protein-protein interaction, and binding analyses. B The diagram shows the number of proteins having each or both [P/A/E]-X-X-S and K-X-X-X-K motifs. C Pathway enrichment of putative substrate proteins of USP7 was analyzed by DAVID analysis. Y-axis indicates the pathway name, and X-axis indicates gene count in the pathways, and the asterisk indicates the p-value (*p < 0.05, **p < 0.01, ***p < 0.001.) D Interaction network between USP7 and putative substrates with high interaction score and with or without the connection revealed. The color of nodes indicates the confirmation of binding between USP7 and proteins (Gray: published and green: nonpublished), and Raf-1 binding with USP7 is shown in purple. Interaction network is shown by Cytoscape. E Endogenous USP7 or Raf-1 was precipitated with an anti-USP7 or an anti-Raf-1 antibody in HeLa cell lysate. F HEK293T cells were transfected with Myc-USP7, and Flag-Raf-1 and immunoprecipitations were performed by an anti-Myc or an anti-Flag antibody. G Purified GST- or GST-USP7 expressed in BL21 was incubated with Flag-Raf-1-overexpressed HEK293T cells. GST- or GST-USP7 was pull downed by GST bead and gel was stained by Coomassie Brilliant Blue. H Immunocytochemical analysis was performed to investigate the localization of… Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Dynamic changes of DNA methylation at different stages of the preimplantation embryo after ELF4 knockdown. (A-C) Immunofluorescence staining of DNA methylation in the control, si-scramble, and si-ELF4 embryos. Scale bars represented 25 µm. (D-F) Semi-quantitative fluorescence intensity analysis of the DNA methylation staining. Different lowercase letters indicate statistical differences (p < 0.05). Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Dynamic changes of DNA methylation at different stages of the preimplantation embryo after ELF4 knockdown. (A-C) Immunofluorescence staining of DNA methylation in the control, si-scramble, and si-ELF4 embryos. Scale bars represented 25 µm. (D-F) Semi-quantitative fluorescence intensity analysis of the DNA methylation staining. Different lowercase letters indicate statistical differences (p < 0.05). Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


Dynamic changes of DNA methylation at different stages of the preimplantation embryo after ELF4 knockdown. (A-C) Immunofluorescence staining of DNA methylation in the control, si-scramble, and si-ELF4 embryos. Scale bars represented 25 µm. (D-F) Semi-quantitative fluorescence intensity analysis of the DNA methylation staining. Different lowercase letters indicate statistical differences (p < 0.05). Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.


The biocompatibility of the BETA scaffold. (a) Scanning electron microscopy (SEM) of the BETA scaffold, which has already been introduced by us elsewhere [21]. The cells were populated onto and into the porous BETA scaffold with alveolar epithelial type II-like A549 cells within 5 days. The scale bars of the SEM images are 50 µm. (b) The chick chorioallantoic membrane (CAM) assay was used to evaluate the biocompatibility of the BETA scaffold. The immunofluorescence (IF) analysis showed the formation of blood vessels on the BETA scaffold characterized by VE-cadherin (red). Cell nuclei are shown in blue (DAPI) and F-actin cytoskeleton in green. (c,d) The CAM-deposited ECM and connective tissue on the membrane analyzed by Masson’s trichrome analysis. (c,d) show the cross-sectioned and diagonally cross-sectioned view of the CAM and BETA scaffold, respectively. Cell nuclei are shown in dark blue/dark brown and connective tissue in green. The scale bars in (b-d) are 100 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/35892691), licensed under a CC BY license.


The IF analysis of the ECM-integrated BETA membrane. (a) the IMR90 human lung fibroblast cells were cultured on the BETA membrane and treated with 5 ng mL−1 TGFβ1. The activated fibroblasts (myofibroblasts) characterized by αSMA (in red) produced high amounts of ECM proteins in response to a fibrotic agent of TGFβ1. (b) The populated scaffolds with IMR90 cells were subsequently decellularized. The selected acellular BETA membrane shows the complete or partial removal of cells retaining collagen I and IV as the two key ECM proteins. Protocol 2 (20 mM NH4OH and 0.1% Triton X-100 for 40 min) with DNAse I treatment (100 U/mL in PBS with Ca2+ and Mg2+) was preferred as the optimized decellularization protocol. Details of the decellularization protocols are shown in Table 1. Collagen I (magenta), collagen IV (red), F-actin cytoskeleton (green), and cell nuclei (DAPI, blue). Scale bars are 100 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/35892691), licensed under a CC BY license.


The IF analysis of the ECM-integrated BETA membrane. (a) the IMR90 human lung fibroblast cells were cultured on the BETA membrane and treated with 5 ng mL−1 TGFβ1. The activated fibroblasts (myofibroblasts) characterized by αSMA (in red) produced high amounts of ECM proteins in response to a fibrotic agent of TGFβ1. (b) The populated scaffolds with IMR90 cells were subsequently decellularized. The selected acellular BETA membrane shows the complete or partial removal of cells retaining collagen I and IV as the two key ECM proteins. Protocol 2 (20 mM NH4OH and 0.1% Triton X-100 for 40 min) with DNAse I treatment (100 U/mL in PBS with Ca2+ and Mg2+) was preferred as the optimized decellularization protocol. Details of the decellularization protocols are shown in Table 1. Collagen I (magenta), collagen IV (red), F-actin cytoskeleton (green), and cell nuclei (DAPI, blue). Scale bars are 100 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/35892691), licensed under a CC BY license.

Product Details

A-11001

Applications

Tested Dilution

Immunohistochemistry (IHC)

Assay-dependent

Immunocytochemistry (ICC/IF)

1 µg/mL

Flow Cytometry (Flow)

1-10 µg/mL

Product Specifications

Species Reactivity

Mouse

Host/Isotype

Goat / IgG

Class

Polyclonal

Type

Secondary Antibody

Immunogen

Gamma Immunoglobins Heavy and Light chains

Conjugate

Alexa Fluor™ 488 Alexa Fluor™ 488 Alexa Fluor™ 488

Excitation/Emission Max

499/520 nm View spectra

Form

Liquid

Concentration

2 mg/mL

Purification

purified

Storage buffer

PBS, pH 7.5

Contains

5mM sodium azide

Storage conditions

4°C, store in dark

Shipping conditions

Ambient (domestic); Wet ice (international)

AB_2534069

Target

IgG

Cross Adsorption

Against human IgG and human serum prior to conjugation

Antibody Form

Whole Antibody

Product Specific Information

To minimize cross-reactivity, these goat anti-mouse IgG whole antibodies have been cross-adsorbed against human IgG and human serum. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there are may be the presence of endogenous immunoglobulins. For a highly cross-adsorbed secondary antibody equivalent, please see product Cat. No. A11029.

Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 488 dye is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 488 dye is pH-insensitive over a wide molar range. Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alexa Fluor 488 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection. The degree of labeling for each conjugate is typically 2-8 fluorophore molecules per IgG molecule; the exact degree of labeling is indicated on the certificate of analysis for each product lot.

Using conjugate solutions: Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step will help eliminate any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining. Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. For the fluorophore-labeled antibodies a final concentration of 1-10 µg/mL should be satisfactory for most immunohistochemistry and flow cytometry applications.

Product will be shipped at Room Temperature.

Target Information

Anti-Mouse secondary antibodies are affinity-purified antibodies with well-characterized specificity for mouse immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488

Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody (A-11001) in IHC