Fig. 3.

a Mouse ESCs were transfected with siRNA-MDM2 or control, and 24 h later treated with MTX 12 h and harvested for Western blot analysis. b, c Mouse ESCs were transfected with siRNA-MDM2, and 24 h later treated with MTX for 12 h. Immunostaining using MDM2 antibodies formed discrete foci and colocalized with H2AK119ub1. Analysis was confined to transfected cells. Error bars represent the SEM. d Mouse ESCs were transfected with siRNA-MDM2 or control, and 24 h later treated with MTX for 12 h. Expressions of the indicated genes were quantified by RT-qPCR. All values were normalized to GAPDH at mRNA level in the same sample. Data are shown as mean (n = 3). *P < 0.05, by Student’s t test. e ChIP assays of MDM2 were performed using mouse ESCs treated with 1 μM MTX for 12 h. Mouse IgG was used as control. Enrichment of NTC-related genes Cdx2, Nestin, Pax6 and Gata4 gene promoters was measured by RT-qPCR. Data are shown as mean (n = 3). *P < 0.05, by Student’s t test. f Mouse ESCs were transfected with siRNA-MDM2 or control, and 24 h later treated with MTX for 12 h. Cells subjected to ChIP analysis with antibodies specific for H2AK19ub1. Immunoprecipitated DNA was quantified by RT-qPCR with primers specific for the 5′ transcribed regions of the indicated genes. Data are shown as mean (n = 3). *P < 0.05, by Student’s t test