DNMTs Play an Important Role in L-ESC Self-Renewal
(A) 2i/L-ESCs were treated with 5-Aza for 3 and 7 days; 2i/L-ESCs retained their typical dome-shaped clonal morphology (results of three independent experiments). Scale bars, 100 μm.
(B) L-ESCs were treated with 5-Aza for 3 and 7 days; a few GOF/GFP+ L-ESCs survived after 7 days of 5-Aza treatment and finally underwent apoptosis (results of three independent experiments). Scale bars, 100 μm.
(C) Left: AP staining on wild-type ESCs and Dnmt3l−/− ESCs switched to L medium for 8 days of culture. Right: quantification of AP+ colonies after 8 days of culture. Error bars are mean ± SD (n = 8). The p values were calculated by two-tailed Student's t test, ∗∗∗p < 0.05.
(D) Relative expression of Dnmt3a by qPCR in Dnmt3a−/− ASCs and Dnmt3a+/+ ASCs. Error bars are mean ± SD (n = 3). The p values were calculated by two-tailed Student's t test, ∗∗∗p < 0.05.
(E) Western blotting analysis for DNMT3A in Dnmt3a−/− ASCs and Dnmt3a+/+ ASCs (results of three independent experiments).
(F) Left: GOF/GFP+ colonies on wild-type ASCs and Dnmt3a−/− ASCs switched to L medium for 10 days of culture. Right: quantification of GOF/GFP+ colonies after 10 days of culture. Error bars are mean ± SD (n = 8). The p values were calculated by two-tailed Student's t test, ∗∗∗p < 0.05.