Fig. 4

Hypermethylation of NANOG and SOX17 Promoters in TET TKO hESCs Leads to a Failure of NANOG and SOX17 Activation Upon hPGC Differentiation. A Scheme of hPGC differentiation rescue in TKO hESCs through NANOG and SOX17 overexpression; B FACS analysis for induction of hPGCs by overexpression of NANOG or/and SOX17 in WT and TKO hESCs without cytokines; C Bright field and fluorescence images of day 4 embryoids upon hPGC induction in B. Scale bar = 200 μm; D RT-qPCR analysis for gene expression during hPGC differentiation in day 4 embryoids by overexpression of NANOG or/and SOX17 in hPGCs induction, the WT day4 hPGCLCs were used as positive control; n = 3 independent experiments. Data are presented as means ± s.d. Statistical analysis was performed by Student’s t-test (two-sided): * represent compared to WT group p < 0.05; E ChIP–qPCR for TET1 in WT and TKO hESCs in NANOG and SOX17 promoters, RPL30 as positive control; n = 3 independent experiments. Data are presented as means ± s.d. Statistical analysis was performed by Student’s t-test (two-sided): *p < 0.05; F Analysis of the percentage of 5mC at the NANOG and SOX17 promoters by Epimark with or without trimethoprim and doxycycline (TD) in day 4 embryoids upon hPGCs induction; n = 3 independent experiments. Data are presented as means ± s.d. Statistical analysis was performed by Student’s t-test (two-sided): * represent compared to WT group p < 0.05