Figure 3.

MEK Inhibition Leads to a Decrease of 5mC Levels and an Increase of 5hmC Levels via DNMT3A/B Reduction and JMJD2C-Mediated TET1 Activation, Respectively

(A) qRT-PCR (n = 4 independent experiments) shows that compared with DMSO control, PD0325901 decreases transcript levels of the Dnmt3 family and Tet1 but does not change Tet2 expression. ESCs were cultured in feeder-free and LIF-free conditions with PD0325901 for 48 hr.

(B) PD0325901 decreases DNMT3A/B protein levels but increases TET1 protein levels in ESCs cultured in feeder-free and LIF-free conditions (n = 3 independent experiments).

(C) Dot blots show that PD0325901 decreases 5mC levels but increases 5hmC levels in ESCs cultured in feeder-free and LIF-free conditions with PD0325901 for 48 hr.

(D) JMJD2C interacts with TET1 and TET2 in a pull-down assay.

(E) JMJD2C associates with TET1 in ESCs in a co-immunoprecipitation experiment.

(F) Jmjd2c-KO ESCs do not express endogenous JMJD2C compared with wild-type ESCs.

(G) PD0325901 and/or CHIR99021 decrease DNMT3B levels, but not H3K9me3 levels, in Jmjd2c KO cells compared with control CCE cells. ESCs were cultured in feeder-free and LIF-free conditions.

(H) Dot blots show that JMJD2C deficiency leads to reduction of 5mC levels by PD0325901 in both control and Jmjd2c-KO ESCs. On the contrary, 5hmC levels are unchanged in Jmjd2c-KO ESCs compared to the control cells.

(I) AP staining shows that the specific JMJD2 inhibitor 5-carboxyl-8-HQ disrupts the pluripotent state induced by PD0325901. Scale bar, 200 μm.

∗∗p < 0.01 and ∗∗∗p < 0.001.