Fig. 5.

a mESCs were cultured in complete medium, complete medium with 1 μM MTX, and supplementary folinic acid (50 mg/L) for 24 h and analyzed by western blot indicated antibody. b Immunostaining assay in mESCs by 1 μM MTX treatment and after folinic acid supplementation. MDM2 antibodies formed discrete foci in mESC cells and colocalized with H2AK119ub1. Analysis was confined to the transfected cells. Error bars represent the SEM. c Expression levels of NTC-related genes Cdx2, Nestin, Pax6 and Gata4 in mESCs cultured in complete medium with either 1 μM MTX for 24 h and folinic acid supplementation or both conditions determined by RT-qPCR. Data are shown as mean (n = 3). *P < 0.05, **P < 0.01, by Student’s t test. d H2AK119ub1 states at representative in NTC-related genes Cdx2, Nestin, Pax6 and Gata4 after folinic acid supplementation compared with MTX treatment alone, as determined by ChIP-qPCR. Data are shown as mean (n = 3). *P < 0.05, by Student’s t test