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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 24 MAR 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 4, pages 693–702, April 2013
How to Cite
Krutá, M., Bálek, L., Hejnová, R., Dobšáková, Z., Eiselleová, L., Matulka, K., Bárta, T., Fojtík, P., Fajkus, J., Hampl, A., Dvořák, P. and Rotrekl, V. (2013), Decrease in Abundance of Apurinic/Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells. STEM CELLS, 31: 693–702. doi: 10.1002/stem.1312
Author contributions: M.K.: collection and assembly of data, data analysis and interpretation, and manuscript editing; L.B., R.H., Z.D., L.E., K.M., T.B., P.F. and J.F.: collection and assembly of data and data analysis and interpretation; A.H. and P.D.: conception and design and financial support; V.R.: conception and design, financial support, collection and assembly of data, data analysis and interpretation, and manuscript writing.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLS EXPRESS January 12, 2013.
- Issue online: 24 MAR 2013
- Version of Record online: 24 MAR 2013
- Manuscript Accepted: 1 DEC 2012
- Manuscript Received: 20 JUL 2012
- Ministry of Health of the Czech Republic. Grant Number: NS10237-3
- Ministry of Education, Youth and Sport of the Czech Republic. Grant Number: MSM0021622430
- European Commission FP6. Grant Number: LSHG-CT-2006-018739
- Grant Agency of the Czech Republic. Grant Number: P302/12/G157
- FNUSA-ICRC. Grant Number: CZ.1.05/1.1.00/02.0123
- European Regional Development Fund
Additional Supporting Information may be found in the online version of this article.
|sc-12-0645_sm_SupplFigure1a.tif||2551K||Supplement Figure 1. An example of a stable karyotypic change in the CCTL14 line visualized using G banding analysis (panel A) and the detail of chromosome change (panel B). Passaging intervals shortened with increasing passage number (panel C, upper graph) as a part of adaptation process. The average passaging interval for each window of 20 passages was plotted. A similar effect on passaging interval was observed for a second, independent cell line, CCTL12 (panel C, bottom graph).|
|sc-12-0645_sm_SupplFigure1b.tif||1319K||Supplement Figure 1b|
|sc-12-0645_sm_SupplFigure1c.tif||2927K||Supplement Figure 1c|
|sc-12-0645_sm_SupplFigure2.tif||2546K||Supplement Figure 2. Telomerase activity as measured by the telomerase reverse transcriptase (TERT) assay in cultured hESCs. The graph shows high and stable TERT activity at passages 32 to 366. The cultured hESCs have much higher TERT activity than both differentiated hESC-derived fibroblasts (hES-F) and embryonic bodies (EBs) derived from them, and than human foreskin fibroblasts (HFF) and mouse embryonic fibroblasts (MEFs). The experiments were done in triplicate, averaged and shown with corresponding standard error bars.|
|sc-12-0645_sm_SupplFigure3.tif||2436K||Supplement Figure 3. Level of NHEJ in hESC nuclear extracts from early medium and late passaged cells. Bars corresponding to the activity of hESC nuclear extracts are labeled according to the passage number of the cells from which the extracts were isolated. Extracts from hESC-derived fibroblasts (hES-Fs), recombinant T4 DNA ligase (Ligase) and sample with no extract added (BL) were included as controls. Activity is expressed as international units (IU). The experiments were done in triplicate, averaged and shown with corresponding standard error bars.|
|sc-12-0645_sm_SupplFigure4.pdf||423K||Supplement Figure 4. Immunohistochemical analysis of APE1 nuclear localization (green) in medium passage hESC line CCTL14. Cells were labeled with mitotracker to visualize mitochondria (red) and DAPI to visualize the nuclei (blue). A colony with heterogeneous expression of APE1 was chosen. White arrows show cells with high and low APE1 expression. No APE1 colocalization with mitochondria or cytosolic localization of APE1 was detected. 100x magnification was used to resolve mitochondria.|
|sc-12-0645_sm_SupplFigure5.tif||1991K||Supplement Figure 5. Immunohistochemical analysis of pluripotency markers in early and late passage hESC line CCTL14. No difference was detected in the level of pluripotency markers NANOG (red) and SSEA3 (green) between early and late passage cells. DAPI (blue) was used to visualize nuclei.|
|sc-12-0645_sm_SupplFigure6a.pdf||440K||Supplement Figure 6. Downregulation of APE1 protein after anti-APE1 siRNA transfection. hESCs were transfected with anti-APE1 siRNA, using X-tremeGene transfection reagent (siAPE1) or transfection reagent alone (X-treme). Cells were fixed and stained 1, 2, or 3 days after transfection (Day 1, Day 2, and Day 3) with anti-APE1 antibody (APE1) and DAPI nuclear staining (DAPI) (Panel A). The third column shows the merged images demonstrating APE1 localization. White bar on the bottom right represents the scale of 200 μm. APE1 protein was detected 3rd day after transfection in hESC CCTL14 by western blot analysis (panel B). Tubulin (Tu) was used as loading control. Expression of APE1 mRNA was analyzed 2nd and 3rd day after the transfection by qRT-PCR (panel C). Transcription levels were plotted relative to tubulin transcription.|
|sc-12-0645_sm_SupplFigure6b.tif||1793K||Supplement Figure 6b|
|sc-12-0645_sm_SupplFigure7a.tif||1383K||Supplement Figure 7. Proliferation and cell cycle parameters of hESC CCTL14 in early passage, late passage and early passage with downregulated APE1. Growth curve (panel 1) was measured after plating 103 cells for total of 91 hours. No difference between APE1 siRNA treated cells (siRNA) and corresponding control (Xtr). Cell cycle distribution of propidium iodide labeled early, late, APE siRNA treated (siRNA) and transfection control (Xtr) hESC CCTL14 was measured using flow cytometry (panel B). While no differences between early hESC, APE1 downregulated cells (siRNA) and corresponding control (Xtr) with strongest G1 phase were observed, more cells in S phase were observed in late passage hESC.|
|sc-12-0645_sm_SupplFigure7b.tif||1728K||Supplement Figure 7b|
|sc-12-0645_sm_SupplFigure8a.tif||594K||Supplement Figure 8. Downregulation of APE1 does not affect γ-H2AX foci formation in non-irradiated cells. Early passage hESCs were transfected with anti-APE1 siRNA (Si) or with transfection reagent alone (EXTR). Control cells of early passage (hESC) and late passage (LATE) were included. γ-H2AX foci were counted using ImageJ 42 and plotted (panel B). Values were for each of five experiments divided by corresponding value of early passage and ploted as relative values together with standard deviation as relative foci per nucleus (panel A). No significant differences were observed between cells with downregulated APE1 and controls. Immunohostochemical analysis of γ-H2AX foci within the course of 20 hours after induction of DNA damage by 3Gy of IR shows dynamics of foci formation and disappearance (panel C). Foci were counted using ImageJ image analysis and plotted (Panel D)|
|sc-12-0645_sm_SupplFigure8b.tif||2820K||Supplement Figure 8b|
|sc-12-0645_sm_SupplFigure8c.pdf||100K||Supplement Figure 8c|
|sc-12-0645_sm_SupplFigure8d.tif||811K||Supplement Figure 8d|
|sc-12-0645_sm_SupplFigure9a.tif||1255K||Supplement Figure 9. Analysis of apoptosis during the early events after DNA damage induction. Presence of apoptotic cells in hESC CCTL14 with downregulated APE1 (siRNA) and corresponding control (Control) was analyzed by TUNEL (green, panel A) for 6 hrs after inducing the DNA damage with 3Gy of IR. DAPI stained nuclei (blue) and phase contrast images of the cells (VIS) are included as control. PARP as an early marker of apoptosis was assayed by western blot (panel B). UV irradiated cells were included as positive control for PARP cleavage (PARP ctrl), tubuline (Tu) as loading control and APE1 as control of siRNA silencing. No change in apoptosis was detected in siRNA treated cells by either method.|
|sc-12-0645_sm_SupplFigure9b.tif||778K||Supplement Figure 9b|
|sc-12-0645_sm_SupplFigure10.pdf||382K||Supplement Figure 10. Methoxamine imhibits γ-H2AX signaling upon IR. Early passage hESC were irradiated with 3Gy after 30 min treatment with 40μM methoxamine. Cells were fixed 60 minutes after IR and stained for presence of phosphorylated histone γ-H2AX. Control cells were not treated with methoxamine. Methoxamine decreases the amount of cells containing γ-H2AX foci in unirradiated (panel A) and number of γ-H2AX foci in irradiated cells (Panel B).|
|sc-12-0645_sm_SupplFigureLegends.pdf||25K||Supplement Figure Legends|
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