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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 19 APR 2011
Copyright © 2011 AlphaMed Press
Volume 29, Issue 5, pages 791–801, May 2011
How to Cite
Yang, P., Wang, Y., Chen, J., Li, H., Kang, L., Zhang, Y., Chen, S., Zhu, B. and Gao, S. (2011), RCOR2 Is a Subunit of the LSD1 Complex That Regulates ESC Property and Substitutes for SOX2 in Reprogramming Somatic Cells to Pluripotency. STEM CELLS, 29: 791–801. doi: 10.1002/stem.634
Author contributions: P.Y. and Y.W.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; J.C., H.L., L.K., Y.Z., and S.C.: provision of study material, data analysis and interpretation; B.Z. and S.G.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. P.Y. and Y.W. contributed equally to this article.
First published online in STEM CELLS EXPRESS March 23, 2011.
Disclosure of potential conflicts of interest is found at the end of this article.
- Issue published online: 19 APR 2011
- Article first published online: 19 APR 2011
- Accepted manuscript online: 23 MAR 2011 04:21PM EST
- Manuscript Accepted: 8 MAR 2011
- Manuscript Received: 20 DEC 2010
Additional Supporting Information may be found in the online version of this article.
|STEM_634_sm_suppinfoFigS1.jpg||636K||Fig. S1. FLAG-RCOR2 stable cell line construction A. Retroviral infection of HeLa cells to generate the cell line stably over-expressing FLAG-RCOR2. The infection efficiency was monitored by GFP reporter gene. BF indicates bright field. B. Sorting of infected HeLa cells that stably expressing FLAG-RCOR2 by flow cytometry. Cells were sorted by GFP expression. The upper panel showed normal HeLa cells, and the lower panel showed sorted infected HeLa cell, in which the ratio of GFP-positive cells is about 29.3%. C. Western blot analysis to confirm the expression of FLAG-RCOR2 in the stable cell line. Western blot with the whole cell extracts from normal HeLa cells (HeLa), infected HeLa cells (unsorted) or infected HeLa cells after sorting (sorted) using antibody against FLAG or β-ACTIN.|
|STEM_634_sm_suppinfoFigS2.jpg||178K||Fig. S2 RT-PCR analysis of Rcor1, Rcor2, Rcor3, Rest and Lsd1 expression in mouse ES cells and MEF cells. Expression of Rcor3 could not be detected in either mouse ES cells or MEF cells. Twenty-five PCR cycles were carried out. Gapdh were used as control.|
|STEM_634_sm_suppinfoFigS3.jpg||1786K||Fig. S3. Charaterization of Rcor2kd mouse ES cells A. The Rcor2kd ES cell colonies were less compact, and the surface of these colonies appeared much rougher. BF indicates bright field. Scale bar 20 μm. B. Rcor2kd ES cells (RFP positive cells) still express OCT3/4 and NANOG, but lower levels of SOX2. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar 20 μm. C. Photographs of the Rcor2kd ES cell-derived chimera mice. Left panel showed the newborn Rcor2kd ES cell-derived chimeric mouse which died within two days was much smaller than the wild type mouse. Right panel showed the photograph of the adult Rcor2kd ES cell-derived chimeric mouse with low chimerism.|
|STEM_634_sm_suppinfoFigS4.jpg||2200K||Fig. S4. Expression profile of Rcor1kd ES cells A. Validation of Rcor1 knockdown efficiency and Lsd1, Rcor2, Rest expression levels by RT-qPCR. Transcript levels were normalized to the levels of mock group. Error bars represent S.D. (n=3). B. RT-qPCR analysis of cell cycle inhibitors including p16Ink4a, p15Ink4b, p21Cip, p53, and p19Arf in Rcor1kd ES cells. Transcript levels were normalized to the levels of mock group. Error bars represent S.D. (n=3) C. RT-qPCR analysis to examine the expression levels of ES marker genes and differentiation marker genes in Rcor1kd ES cells. Transcript levels were normalized to the levels of mock group. Error bars represent S.D. (n=3). D. RT-qPCR analysis of genes involved in different signaling pathways, epithelial genes and mesenchymal genes in Rcor1kd ES cells. Transcript levels were normalized to the levels of mock group. Error bars represent S.D. (n=3).|
|STEM_634_sm_suppinfoFigS5.jpg||325K||Fig. S5.Validation of p16Ink4a knockdown efficiency by RT-qPCR in MEF cells. The siRNA specific for p19Arf was used to test the siRNAs/ specificity. Transcript levels were normalized to those of the mock group that transfected with negative control siRNA. Error bars represent S.D. (n=3).|
|STEM_634_sm_suppinfoFigS6.jpg||653K||Fig. S6. Validation of Rcor2 and Rcor1 knockdown or over-expression during iPS induction. Both Rcor2 and Rcor1 expression levels were monitored by RT-qPCR (day 8 and 12) to confirm the knockdown and overexpression during somatic reprogramming. The expression levels were normalized to those of MEF cells.|
|STEM_634_sm_suppinfoFigS7.jpg||311K||Fig. S7. Rcor2 can substitute Sox2 in iPS generation and is required for reprogramming efficiency. 5×104 MEF cells isolated from rtTA-OG2 transgenic mice were used in iPS induction. GFP+ colonies was counted after culturing these cells in ES cell medium supplemented with 1 μg/mL doxycycline for 20 days. The values are represented as mean and S.D. of the averages of three independent experiments. Rcor2 but not Rcor1 can increase the number of GFP+ colonies. OKMR2 or OKR2 combination is sufficient for iPS induction though less efficient.|
|STEM_634_sm_suppinfoFigS8.jpg||890K||Fig. S8. Rcor2 and Lsd1 target T/Brachyury and p16 in mouse ESCs. A. ChIP-qPCR analyses of RCOR2 binding at selected target genes in mouse ES cells. B. ChIP-qPCR analyses of LSD1 binding at selected target genes in mouse ES cells.|
|STEM_634_sm_suppinfoFigS9.jpg||644K||Fig. S9. Comparison of the global gene-expression profiles between Rcor2kd and mouse R1 ES cells A. Pearson correlation analysis of probes was performed to cluster R1 ES cells and Rcor2kd ES cells. Red indicates increased expression compared to median levels of the four samples, whereas green means decreased expression. B. Expression levels of ES-specific and pluripotency-associated genes in R1 ES cells and Rcor2kd ES cells. Error bars represent S.D. (n=3).|
|STEM_634_sm_suppinfoTable1.xls||32K||Supporting Information Table 1|
|STEM_634_sm_suppinfoTable2.xls||25K||Supporting Information Table 2|
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