Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells
Abstract
:1. Introduction
2. Results
2.1. hASC Isolation and Characterization
2.2. Knockdown of NANOG Silencing by Lentivirus
2.3. Analysis of PREF1
2.4. Downregulation of NANOG Inhibited Cell Proliferation, Increased Population Doubling Time and Reduced Differentiation Potential
2.5. Knockdown of NANOG Inhibited the Expression of CCND1/Cycline D1, Enhanced the Expression of p21 and p27 and Induced Cell Cycle Block in the G0/G1 Phase
2.6. Knockdown of NANOG Regulates DNMT1
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Cell Infection with Lentiviral Particles
4.2. RNA Isolation and Quantitative RT-PCR
4.3. Western Blot Analysis
4.4. Analysis of Cell Cycle Status of MSCs with Lentivirus
4.5. Colony-Forming Assay
4.6. Population Doubling and Cell Proliferation Curve
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
MSC | Mesenchymal Stem Cell |
hASC | human Adipose Stem Cell |
p21/CDKN1A | Cyclin-dependent kinase inhibitor 1A |
p27/CDKN1B | Cyclin-dependent kinase inhibitor 1B |
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Primary Antibody/Localization Marker | Code Number | Diluition | Incubation |
---|---|---|---|
NANOG, nuclear and cytoplasmatic | sc-293121, Santa Cruz Biotechnology | 1:500 | o/n, 4 °C |
DNMT1, nuclear | sc-271729, Santa Cruz Biotechnology | 1:500 | o/n, 4 °C |
B-Actin clone AC-74 | A5316, Sigma Aldrich | 1:10,000 | o/n, 4 °C |
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Pitrone, M.; Pizzolanti, G.; Coppola, A.; Tomasello, L.; Martorana, S.; Pantuso, G.; Giordano, C. Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells. Int. J. Mol. Sci. 2019, 20, 2580. https://doi.org/10.3390/ijms20102580
Pitrone M, Pizzolanti G, Coppola A, Tomasello L, Martorana S, Pantuso G, Giordano C. Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells. International Journal of Molecular Sciences. 2019; 20(10):2580. https://doi.org/10.3390/ijms20102580
Chicago/Turabian StylePitrone, Maria, Giuseppe Pizzolanti, Antonina Coppola, Laura Tomasello, Stefania Martorana, Gianni Pantuso, and Carla Giordano. 2019. "Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells" International Journal of Molecular Sciences 20, no. 10: 2580. https://doi.org/10.3390/ijms20102580