Depletion of EZH2 results in myogenic differentiation of embryonal RD cells in growth medium (GM). RD cells were transfected (t0) with EZH2 siRNA or control (CTR) siRNA and after 24 h silenced again. They were cultured in proliferating growth medium (GM, i.e. supplemented with 10% of fetal calf serum) for the following experimental procedures. (a) RD cells were analyzed for the induction of muscle-like differentiation 6 days post-siRNA transfection. Representative immunofluorescence showing de novo expression of endogenous Myosin Heavy Chain (MHC, red) in multinucleated fibers of EZH2 siRNA-transfected cells. DAPI was used for nuclear staining. Representative of four assays. (b) Western blot showing de novo expression of Troponin I 6 days post-siRNA transfection. GAPDH served as loading control. (c) Western blot showing EZH2, p21Cip1, Myogenin and GAPDH expression in RD cells 48 h and 72 h after EZH2 or CTR siRNA transfection and in untreated RD cells. (*band: longer exposure). Representative of four independent experiments. GAPDH served as loading control. (d) mRNA levels (real time qRT-PCR) of Myogenin, MCK, and p21Cip1 in RD cells 48 h and 72 h after EZH2 siRNA treatment were normalized to GAPDH levels and expressed as fold increase over untreated condition (1 arbitrary unit, not reported). Columns, means; Bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test). (e) ChIP assays on RD cells 72 h after EZH2 or CTR siRNA transfection showing the recruitment of EZH2 and the levels of histone H3 trimethylation on Lys27 (H3K27me3) on Myogenin, MCK, MHC and SMAD6 (as negative control) regulatory regions. Normal rabbit IgG were used as negative control. Graphs represent the percent of immunoprecipitated material relative to input DNA. Results are the average of three independent experiments. *P <0.05 (Student’s t-test).