MC3T3 cells were transfected with control and Ezh2 siRNAs on day zero and osteogenic differentiation was started on the same day. On day two, cells were treated with vehicle, GSK126 (5μM) or EPZ-6438 (5μM). Vehicle and drugs were removed on day five. (A) Western blot analysis of histone marks and reference proteins collected at day two and five of differentiation. Alizarin red staining (B) and quantification (C) was conducted on day 24 of differentiation (n = 3, mean ± STD). (D) RT-qPCR of Ezh2 and osteogenic markers at day five and ten (n = 3, mean ± STD). Statistical significance to siCtrl + Vehicle control group indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. G, GSK126; E, EPZ-6438.