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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 20 AUG 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 9, pages 1901–1910, September 2012
How to Cite
Hyka-Nouspikel, N., Desmarais, J., Gokhale, P. J., Jones, M., Meuth, M., Andrews, P. W. and Nouspikel, T. (2012), Deficient DNA Damage Response and Cell Cycle Checkpoints Lead to Accumulation of Point Mutations in Human Embryonic Stem Cells. STEM CELLS, 30: 1901–1910. doi: 10.1002/stem.1177
Author contributions: N.H.-N.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; T.N.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; M.M. and P.W.A.: financial support, provision of study materials, data analysis and interpretation, and final approval of manuscript; J.D.: provision of study materials, collection and/or assembly of data, and final approval of manuscript; P.J.G. and M.J.: collection and/or assembly of data, data analysis and interpretation, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS July 20, 2012.
- Issue published online: 20 AUG 2012
- Article first published online: 20 AUG 2012
- Accepted manuscript online: 20 JUL 2012 09:48AM EST
- Manuscript Accepted: 21 MAY 2012
- Manuscript Received: 23 JAN 2012
- Yorkshire Cancer Research. Grant Numbers: PP005, S298
- U.K. Medical Research Council. Grant Number: G0700785
- fellowship from the Fonds de recherches du Québec—Santé
Additional Supporting Information may be found in the online version of this article.
|SC-12-0078_sm_SupplFigure1.tif||2436K||Figure S1. 1. UV-;induced apoptosis in hESCs H7 cells were irradiated with 5 J/m2 UV and harvested after 3, 6 and 24 hours. Floaters were harvested separately from adherent cells at 6 and 24 hours (there were too few floaters for analysis at 3 hrs). Cells, excluding debris, were stained with Annexin V (xaxis) and 7-;AAD (y-;axis) and analyzed by FACS. Quadrants: LL, healthy cells, LR: early apoptosis, UR: late apoptosis. See synthetic graph in figure 1A.|
|SC-12-0078_sm_SupplFigure2.tif||563K||Figure S2. Cell cycle progression in UV-;irradiated hESCs Shef5 cells were irradiated with 0, 5, 7.5 or 10 J/m2 UV and harvested 24 hours later, after a 4-;hour labelling with BrdU, and 48 hours later after overnight labelling with BrdU. Cells were stained with propidium iodide and anti-;BrdU antibodies and analyzed by FACS. See figure 2 for BrdU versus propidium iodide graphs.|
|SC-12-0078_sm_SupplFigure3.tif||1004K||Figure S3. UV-;irradiation of hESCs pulse-;labelled with BrdU A: Schematic representation of the experimental protocol: Shef5 cells were pulse-;labelled 20 min with 20 μM BrdU, chased 5 min with thymidine, and harvested (small arrow heads) at various times after labelling (“No UV” samples). At the same time points, two additional aliquots of cells were irradiated with 5 J/m2 UV light (purple arrows) and cultured for another 3 and 6 hours. B: Progression of BrdU-;labelled cells through the cell cycle. Cells were stained with 7-; AAD, anti-;BrdU and anti-;cleaved caspase 3 antibodies, and analyzed by FACS, excluding clumps and sub-;G1 cells. The graph presents the distribution of BrdU-;labelled cells between G1/Early-;S phase and Late-;S/G2 phase, based on DNA content. C: UV-;inducted apoptosis in BrdU-;labelled cells. The amount of caspase 3 cleavage (see also Fig. S9B) in BrdU-;positive cells was appraised by FACS. The various curves correspond to the time of irradiation, with respect to BrdU labelling (same symbols as in panel B). The shaded area shows the range of values observed in the asynchronous, BrdU-;negative cells. D: Raw data. DNA content (x-;axis) versus cell number, for the total population (red), or BrdU-;labelled cells (green).|
|SC-12-0078_sm_SupplFigure4.tif||95K||Figure S4. p21/Waf1 mRNA in UV-;irradiated hESCs Shef5 cells were irradiated (or not) with 5 J/m2 UV and RNA was isolated at various time points. The amount of p21 mRNA was quantified by real-;time RT-;PCR, and normalized against U1 mRNA by the delta-;delta Ct method, taking cycle efficiency into account. Results are displayed as percent of the positive control: HCT116 cells challenged with 10 mM butyrate.|
|SC-12-0078_sm_SupplFigure5.tif||460K||Figure S5. Cell surface marker analysis in UV-;irradiated hESCs H7 cells were irradiated (or not) with 5 J/m2 UV and analyzed by FACS 24 hours later for surface markers SSEA3, SSEA4, TRA-;1-;60, and TRA-;1-;81 (not shown). X-;axis: forward scatter, Y-;axis: antibody signal.|
|SC-12-0078_sm_SupplFigure6.tif||136K||Figure S6. Clonogenic potential of UV-;irradiated hESCs H7 cells were irradiated (or not) with 5 J/m2 UV, dissociated 5 days later, plated as single cells at various densities, and cultured for one week. Cells were then fixed and stained with the DNA dye Hoechst 33258. Colonies (top) and cells (bottom) were counted with an automated scanning microscopy system.|
|SC-12-0078_sm_SupplFigure7.pdf||113K||Figure S7. Differentiation of UV-;irradiated hESCs H7 cells were irradiated with 5 J/m2 UV and cultured for 4 days. To produce embryoid bodies, cells were treated with collagenase, scrapped into large clumps, and cultured in non-;adherent petris for 5 days, then plated in gelatinized 6-;well plates and cultured for another 10 days. Cells were then fixed, strained with antibodies against the following proteins: beta-;tubulin (ectoderm), smooth muscle actin (mesoderm) and alphafoetoprotein (endoderm), and counter-;stained with Hoechst 33258.|
|SC-12-0078_sm_SupplFigure8.tif||1897K||Figure S8. Induction of a UV-;resistance phenotype in hESCs A: H7 cells were irradiated (or not) with 5 J/m2 UV, aliquots were harvested 3, 6, and 24 hours, stained with Annexin V, propidium iodide and anti-;SSEA3 antibodies, and analyzed by FACS. The remainder of irradiated cells were allowed 1 week to recover (with a passage at day 4), then re-;irradiated with 5 J/m2 UV and analyzed as above. Results are presented separately for SSEA3-;negative and SSEA3-;positive cells. See figure 4A for a synthetic representation. B,C: Similar results were obtained with Shef5 cells, although it took an additional cycle of UV-;irradiation to obtain the levels of resistance observed in H7. ***: significantly different (p<0.001) from first irradiation by both Kolmogorov-;Smirnov and Probability Binning tests (FlowJo).|
|SC-12-0078_sm_SupplFigure9.tif||255K||Figure S9. DDB1 and DDB2 expression, and caspase 3 cleavage in hESCs. A: Shef5 cells were irradiated with 5 J/m2, whole cell extracts were prepared as in figure 3, and analyzed by Western blotting for the presence of DDB1 and DDB2/XPE. HeLa extracts shown for comparison. B: Same samples as above, analyzed for the presence of the 17 kDa cleaved fragment of activated caspase 3.|
|SC-12-0078_sm_SupplFigure10.tif||314K||Figure S10. PCNA ubiquitination in UV-;irradiated hESCs Shef5 cells were irradiated with 5 J/m2 and whole cell extracts were prepared as in figure 3. Western blots were probed with antibodies against PCNA and beta-;actin. The PCNA blot was overexposed (top) to reveal the slower-;migrating ubiquitinated PCNA (arrow). Bottom graph: amount of ubiquitinated PCNA, normalized against beta-;actin.|
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