Extended Data Fig. 1: Pluripotency identification of iPSCs derived from senescent somatic cells.
From: Glis1 facilitates induction of pluripotency via an epigenome–metabolome–epigenome signalling cascade
a, OG2 MEFs were infected with retroviral SKO plus Flag or Glis1. GFP+ colonies were counted on day12, day14, day16, day18, day20. Data are presented as the mean ± S.D. (n = 3, each group).Group differences are analyzed by the two-tailed Student’s t test. b, OG2 MEFs were infected with retroviral SKO plus Flag or Glis1. (left): GFP colony pictures on day35 during reprogramming; (right): GFP+ colonies were counted on the indicated days during reprogramming. Data are presented as the mean ± S.D. (n = 3, each group).Group differences are analyzed by the two-tailed Student’s t test. c, Bright and β-gal staining images of P2-MEFs, P7-MEFs, P8-Flag-MEFs and P8-Glis1 expressed stably MEFs (P8-Glis1-MEFs) (a senescent cell was circled in red in MEFs). Scale bar: 100 μm. One replicate (n = 1, each group) was used for bright and β-gal staining images analysis. n = 3 independent experiments were repeated with similar results (a–b). d, β-gal positive cells counting for P2-MEFs, P7- MEFs (left); P8-Flag-MEFs, P8-Glis1-MEFs (right). Data are presented as the mean ± S.D. (P2-MEFs, n=52; P7- MEFs, n=58; P8-Flag-MEFs, n=58; P8-Glis1-MEFs, n=75 fields, each group).Group differences are analyzed by the two-tailed Student’s t test. e, Cell proliferation capacity analysis of P2-MEFs, P7-MEFs (left); P8-Flag-MEFs, P8-Glis1-MEFs (right) by Edu. Data are presented as the mean ± S.D. (n = 3, each group).Group differences are analyzed by the two-tailed Student’s t test. f, RT-qPCR analysis for detecting the expression of p16, p21 and p53 in P8-Flag-MEFs or P8-Glis1-MEFs. Data are presented as the mean ± S.D. (n = 3, each group).Group differences are analyzed by the two-tailed Student’s t test. g, Western blot analysis of P16, P21 and P53 upon Glis1 overexpression in P8-Flag-MEFs or P8-Glis1-MEFs (n = 1 independent experiments). h, Oct4-GFP images and immunofluorescence staining for the indicated pluripotency markers (red) for P7-iPSCs and P8-iPSCs clones derived from OG2 MEFs (n = 6 clonies for each group). Scale bar: 50 μm (left); RT-qPCR showing endogenous expression of indicated pluripotency genes in MEFs, mouse embryonic stem cells (R1) and P7-iPSCs and P8-iPSCs clones (right).
