Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cloning of Inducible HMGB-shRNA Constructs
2.2. Generation of Stably Transfected hESCs with Inducible HMGB-shRNA Constructs
2.3. Human Neuroectodermal Lineage Commitment from hESCs
2.4. Crystal Violet Staining
2.5. Western Blotting
2.6. Cell Cycle Assay by Flow Cytometry
2.7. Immunocytochemistry
2.8. Confocal Microscopy
2.9. Annexin V-FITC/Propidium Iodide Double-Staining Assay
2.10. Cell Cycle and Apoptosis of Etoposide-Treated Cells
2.11. Image Acquisition and Analysis
2.12. Determination of Telomerase Activity by TRAP Assay
2.13. Statistical Analysis
3. Results
3.1. Pluripotent hESCs with Downregulated HMGB1/2 Expression Retain Stemness and Pluripotent State
3.2. Distinct Impact of HMGB1/2 KD on Survival and Neuroectodermal Lineage Commitment
3.3. Pluripotent hESCs Exposed to Etoposide are more Susceptible to Cell Death than the hESC-Derived Progenitor Cells
3.4. Cell Cycle Progression in HMGB KD hESCs and hNECs Treated with Etoposide
3.5. Apoptosis of Etoposide-Treated hESCs is Enhanced upon HMGB1 KD
3.6. The Impact of HMGB1/2 Knockdown on Apoptosis of Etoposide-Treated Progenitor Cells Depends on the Differentiation Protocol of hESCs
3.7. HMGB1 KD Promotes Cell Death Induction in Etoposide-Treated hESCs and hNECs via Activation of Caspase-3 Pathway
3.8. Impact of HMGB1/2 KD on Telomere Integrity is Distinct in Etoposide-Treated hESCs and hNECs
3.9. The Distinct Impact of HMGB2 KD on Telomerase Activity in Etoposide-Treated hESCs and hNECs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Bagherpoor, A.J.; Kučírek, M.; Fedr, R.; Sani, S.A.; Štros, M. Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide. Biomolecules 2020, 10, 1450. https://doi.org/10.3390/biom10101450
Bagherpoor AJ, Kučírek M, Fedr R, Sani SA, Štros M. Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide. Biomolecules. 2020; 10(10):1450. https://doi.org/10.3390/biom10101450
Chicago/Turabian StyleBagherpoor, Alireza Jian, Martin Kučírek, Radek Fedr, Soodabeh Abbasi Sani, and Michal Štros. 2020. "Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide" Biomolecules 10, no. 10: 1450. https://doi.org/10.3390/biom10101450