Characterization of early neuronal differentiation markers in precursor cell lines using multiplexed luciferase reporters and immunofluorescence imaging

by Douglas E. Hughes, Ph.D.; Angela M. Schoolmeesters, B.S.1; Strato Katsoulidis, Ph.D.; Jae Choi, Ph.D.; Janaki Narahari, Ph.D.; Suk Hong, Ph.D.; Surbhi Desai, Ph.D.; Melissa L. Kelley, Ph.D.1; Brian Webb, Ph.D.; - 03/08/13

Cellular differentiation studies require sensitive tools to detect subtle phenotypic changes in cellular signaling pathways and morphology. In the present study, we used luciferase assays and high-content immunofluorescence cellular imaging to monitor phenotypic changes in neuronal precursor cell lines following retinoic acid-induced differentiation.

Retinoic Acid (RA), the biologically active form of Vitamin A, is widely known to drive the differentiation of numerous precursor cell lines. RA functions through binding to nuclear receptors and inducing transcription of specific target genes. Neuronal differentiation requires down-regulation of stem cell-specific genes and up-regulation of lineage-specific genes (Figure 1). Once retinoids are bound by the RAR/RXR heterodimer they become transcriptionally activated. As a consequence they are degraded by the proteasome. Phosphorylation in the activation domain one (AF1) and presence of the activation domain 2 (AF2) trigger the ubiquitination of the receptors. Moreover, heterodimerization of RAR and RXR accelerate degradation of the receptors. Suppressor of zeste 12 (SUZ12) protein is a member of the Polycomb group (PcG) family of transcriptional repressors. The SUZ12 protein plays a role in the suppression of lineage-specific genes and maintains pluripotency of stem cells. Previous studies have shown that SUZ12 mRNA decreases in stem cell populations upon lineage specific differentiation, suggesting this gene is primarily regulated at the transcriptional level. For this reason, we hypothesized that this promoter would make an excellent marker for cellular differentiation.

A13n02-Fig1Figure 1. Biology of neuronal differentiation. Neuronal differentiation requires down-regulation of stem cell-specific genes and up-regulation of lineage-specific genes.

To validate our retinoic acid induced cellular differentiation systems, we used immunofluorescence imaging to monitor changes in stem cell-specific markers such as SUZ12, Nanog, Sox-2, and SSEA-3, as well as lineage specific markers SSEA1, MAP2, and beta-III-tubulin in three cell types.

To examine transcriptional decrease in suppressor of zeste 12 (SUZ12) during differentiation, we utilized multiplexed luciferase assays to measure SUZ12 promoter activity and cellular response to retinoic acid. In addition, we treated cells with siRNA to decrease the levels of key transcription factors thought to regulate SUZ12 transcription. To measure changes in the promoter activity of theSUZ12 gene, we cloned the -0.5 kb genomic promoter region of the human SUZ12 gene upstream of Cypridina luciferase. We hypothesized that Cypridina luciferase activity would decrease in response to RA induced cellular differentiation. Using siRNA-mediated knockdown, we sought to mimic the decrease in SUZ12 promoter activity in the absence of RA. To monitor cellular response to retinoic acid, we cloned retinoic acid response elements (RAREs) upstream of Gaussia luciferase for use in multiplex luciferase assays.

Results and discussion

Our results (Figures 2 to 7) demonstrate the utility of multiplex luciferase assays as an early monitoring system for cellular differentiation and the effects of siRNA mediated protein knockdown. In addition, we observed down-regulation of the stem cell markers Nanog and Sox-2 and up-regulation of differentiation-specific markers SSEA1 and beta-III-tubulin by immunofluorescence imaging.

A13n02-Fig2Figure 2. Validation of RA induced differentiation in NCCIT and SH-SY5Y cells using immunofluorescence (IF) imaging. NCCIT cells treated with RA show a decrease in stem cell markers Nanog, SSEA-3, and TRA-1-60 (left panel). In addition, a decrease in SOX2 and an increase in the somatic marker SSEA-1 and neuronal marker beta-III-tubulin is also observed (data not shown). SH-SY5Y cells treated with RA show a decrease in the stem cell specific protein SUZ12 and an increase in neuronal markers beta-III-tubulin and MAP2 (right panel). Antibody staining is shown in green, F-Actin in red, and nuclei in blue.
A13n02-Fig3Figure 3. Quantitative analysis of IF data in NCCIT and SH-SY5Y cells. There is a significant decrease in the SUZ12 expressing cell population upon RA treatment (RA) compared to untreated (no RA).
A13n02-Fig4Figure 4. Alignment of the SUZ12 proximal promoter region from human (-0.5 kb hSUZ12) and mouse (-0.5 kb mSUZ12). There is > 96% homology at the DNA level. In addition, evolutionary conservation of transcription factor binding sites, shown in red, for transcription factors important in stem cell maintenance, suggests they may regulate the transcription of the SUZ12 gene.
A13n02-Fig5Figure 5. Design of triple reporter luciferase assays. Cypridina signal are measured in the cell medium using the Cypridina Luciferase Flash Assay Kit, and Gaussia and Red Firefly signals are measured in the cell lysate using the Gaussia-Firefly Luciferase Dual Assay Kit. Alternatively, Cypridina and Gaussia signals are measured in the cell medium using Cypridina Luciferase Flash Assay Kit or Gaussia Luciferase Flash Assay Kit, and Red Firefly signals are measured in the cell lysate using the Firefly Luciferase Flash Assay Kit.
A13n02-Fig6Figure 6. SUZ12 promoter activity decreases, and RAR element activity increases, as expected, in response to retinoic acid (RA) treatment. NCCIT, SH-SY5Y, or Neuroscreen-1 cells were co-transfected with SUZ12-Cypridina, RAR-Gaussia, and CMV-Red Firefly plasmids. 24 hours later, cells were treated with RA for 72 hours. Luciferase assays were performed for each respective luciferase and Cypridina and Gaussia signals were normalized to the Red Firefly signal. Results are displayed as fold induction or repression relative to the untreated condition (no RA).
A13n02-Fig7Figure 7. SUZ12-Cypridina Luciferase expression decreases after knockdown of transcription factors by siRNA in the absence of RA treatment. SH-SY5Y cells were co-transfected with 100nM of the indicated siRNA and 50ng each of SUZ12-Cypridina and CMV-Red Firefly plasmid. Luciferase flash assays were performed for each respective luciferase and Cypridina signals were normalized to the Red Firefly signal. Results are displayed as fold induction or repression relative to the Non-targeting Control siRNA (NTC). Starred bars have p-value < 0.05.
  • Immunofluorescence imaging of RA treated NCCIT and SH-SY5Y cells reveals decreases in differentiation-specific markers Nanog and Sox-2 and increases in the somatic markerSSEA1 and neuronal marker beta-III-tubulin.
  • RA treatment of NCCIT, SH-SY5Y and NS-1 cells decreases the stem cell-specific markerSUZ12 likely through transcriptional repression mechanism.
  • GATA-1, MZF1, and EP300 siRNA-mediated knockdown mimics the effects of RA on theSUZ12 promoter.
  • The SUZ12 promoter contains potential binding sites for GATA-1MZF1, and EP300suggesting that RA treatment may affect the levels of these transcription factors.
  • Multiplex luciferase assays are a successful method for early monitoring of cellular differentiation and the effects of siRNA mediated protein knockdown.

Plasmid construction: The RARE-Gaussia Luc reporter plasmid was constructed by cloning three copies of the retinoic acid response element plus minimal promoter into the Thermo Scientific Pierce pMCS-Gaussia Luc Reporter Plasmid (Part No. 16146). The -0.5kb SUZ12-Cypridina Luc plasmid was constructed by cloning a -0.5kb fragment of the SUZ12promoter PCR amplified from a region immediately 5′ of the transcription start site into the Thermo Scientific Pierce pMCS-Cypridina Luc plasmid (Part No. 16149). The Thermo Scientific Pierce pCMV-Red Firefly Luc plasmid (Part No.16156) was used as a normalization control.

Cell culture: All cell lines were maintained at 37°C with 5% carbon dioxide in a humidified cell culture incubator. NCCIT cells (ATCC Cat# CRL-2073) cells were cultured in ATCC-formulated RPMI-1640 Medium with 10% fetal bovine serum (complete). Cells were passaged every two days and maintained at 50-70% confluence. SH-SY5Y (ATCC Cat# CRL-2266) cells were maintained in 1:1 mixture of DMEM and Hams F12 Nutrient Medium, supplemented with 10% Fetal Bovine Serum, 2nM L-Glutamine, 0.15% Sodium Bicarbonate, 1nM Sodium Pyruvate, and 100nM Non-Essential Amino Acids. Neuroscreen-1 (Thermo Scientific) cells, a PC-12 subclone, were maintained in RPMI 1640 Medium, supplemented with 5% Fetal Bovine Serum and 10% Equine Serum and seeded on Collagen Type-I Rat Tail coated flasks. Thawing and passaging of cell lines was performed according to supplier’s recommendations.

Transfection: NCCIT cells were seeded at 10,000 cells per well in 96-well tissue culture plates the day before transfection in maintenance medium. The three described plasmid constructs (50/50/50ng) were diluted in 10μL of serum-free medium. Thermo Scientific TurboFect Transfection Reagent (0.3μL) (Part No. R0533) was added to the diluted DNA and mixed by pipetting. The mixture was added drop-wise to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours prior to treatment. SH-SY5Y and Neuroscreen-1 cells were seeded at 20,000 cells per well in 96-well tissue culture plates, collagen-I coated for Neuroscreen-1 cells, the day before transfection in maintenance medium. Thermo Scientific DharmaFECT Duo Transfection Reagent (Cat# T-2010) was utilized to deliver siRNA and plasmid simultaneously. Three different luciferase constructs were co-transfected with the siRNA pools. Transfection reagent, siRNA, and the DNA were complexed in MEM-RS Medium for 20 minutes before adding to the cells per the manufacturer’s protocol. SH-SY5Y cells were transfected with 0.3µL of DharmFECT Duo per well and 0.4µL for Neuroscreen-1 (NS-1) cells. In all transfections, the final concentration of siRNA was 100nM and 50ng of each plasmid. Thermo Scientific ON-TARGETplus SMARTpool siRNA reagents: GATA1 #J-009610, NFκB1 #J-003520, EP300 #J-003486, MZF1 #J-006578, and Non-targeting Pool #D-001810-10. Cells were either assayed 72 hours post-transfection for siRNA experiments or treated with retinoic acid 24 hours post-transfection and assayed 72 hours after treatment.

Treatment and sample collection: The medium was replaced with complete medium containing (treatment) or not containing (control) all-trans retinoic acid (ATRA; Sigma Cat #R2625) at a concentration of 10µM in DMSO. Following incubation for 72 hours, medium was collected for measurement of activity. The cells were then lysed with 100μL of Thermo Scientific Pierce Luciferase Cell Lysis Buffer (Part No. 16189). Either the collected medium or lysates were used for luciferase activity measurement.

Luciferase reporter assays: For NCCIT cells, the Gaussia activity was determined using the Thermo Scientific PierceGaussia Luciferase Flash Assay Kit (Part No. 16158). The Cypridina and Red Firefly activity was determined using the Thermo Scientific Pierce Cypridina-Firefly Luciferase Dual Assay Kit (Part No. 16184). Bioluminescence signals (RLUs) were detected using a Thermo Scientific Varioskan Flash Luminometer equipped with reagent injectors (Signal integration time = 1 second). Gaussia and Cypridina signals were then normalized to the Red Firefly signal. SH-SY5Y and NS-1 cells were assayed at 72 hours post-transfection (siRNA experiments) or 96 hours post-transfection (retinoic acid treated experiments). Thermo Scientific Pierce Luciferase Flash Assay Kits were used to determine Luciferase expression levels on the EnVision* Multilabel Reader. Twenty µL of medium from the cell plates were transferred to Thermo Scientific Nunc plates (Fisher Scientific Cat# 136102) and performed in the Gaussia (Part No. 16159) andCypridina (Part No. 16169) Flash Luciferase Assays per the supplier’s protocol. Cells were washed with DPBS and lysed in 70µL of Cell Lysis Buffer. Twenty µL of cell lysate was transferred to Nunc plates and ran in the Firefly Luciferase Flash Assay (Part No. 16175) according to the protocol. Firefly Luciferase expression was used for transfection normalization of Cypridina and Gaussia expression. Non-targeting Pool (NTC) was used for relative normalization purposes in the siRNA experiments.

Immunofluorescence imaging: Cells were seeded at either 10,000 or 20,000 cells per well in 96-well tissue culture plates in maintenance medium. Retinoic acid was added to the cells the following day. Cells were fixed in 4% paraformaldehyde 72 hours post-treatment. Cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Part No. 37525) for 15 minutes at room temperature. Cells were then probed with antibodies against MAP2 (Cat# 1861751), beta-III-tubulin (Cat# 1860254), Nanog (Ab No. PA1-097), SSEA-3 (Ab No. MA1-020), TRA-1-60 (Ab No. MA1-023), SSEA-1 (Ab No. MA1-022), SOX-2 (Ab No. PA1-094) and SUZ12 (Cell Signaling Technology, Cat# 3737S) at dilutions of 1:50-1:100 for at least 1 hour at room temperature shown in green. Cells were washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG (Part No. 35502), goat anti-rabbit IgG (Part No. 35503) or fluor-labeled IgM secondary antibodies at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 phalloidin (Part No. 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Part No. 62249). Images were taken on a Thermo Scientific ArrayScan or ToxInsight Imager at 20X magnification.

Quantitative image analysis: Quantitative analysis was performed using the Thermo Scientific Cellomics vHCS software and the appropriate assay parameters for cellular compartment analysis and neuronal profiling. Representative wells from treated and untreated SH-SY5Y and NCCIT cell populations were analyzed for average signal intensity. Nuclear staining with Hoechst dye (Part No. 62249) was used for cell identification and quantification.

General references
  • Nakajima, Y., et al. (2004). cDNA Cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca. Biosci. Biotechnol. Biochem. 68(3):565-570.
  • Pasini D., et al. (2007). The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation. Mol Cell Biol. 27(10): 3769–3779.
  • Rohwedel J, Guan K, Wobus AM. (1999). Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs. 165(3-4):190-202.
  • Shimomura, O. (2006). Bioluminescence: Chemical Principles and Methods. World Scientific Publishing Co. Pte. Ltd, Hakensack , NJ.
  • Szent-Gyorgyi, C., et al. (1999). Cloning and characterization of new bioluminescent proteins. Part of the SPIE Conference on Molecular Imaging: Reporters, Dyes, Markers, and Instrumentation. San Jose, CA. Proc. SPIE 3600:4-11.
  • Tannous, B. A., et al. (2005). Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Molecular Therapy 11:435-443.
Editor's note

These data were originally presented in the following poster:

Hughes, D., et al. (2012). Characterization of early phenotypic changes in differentiating NCCIT cells using multiplexed luciferase reporters and immunofluorescence imaging. Poster #633.25. Presented Tuesday, October 16, 2012. Society for Neuroscience Annual Meeting. New Orleans, LA.

For Research Use Only. Not for use in diagnostic procedures.