EZH2 depletion inhibits embryonal rhabdomyosarcoma (RMS) cell proliferation. (a) Western blot showing EZH2 and β-actin (loading control) in whole-cell lysates from embryonal RMS cell lines and normal human myoblasts SKMC as control, all cultured in proliferating growth medium (GM, i.e., supplemented with 10% fetal calf serum). EZH2* band: longer exposition. Representative of three independent experiments. (b) Western blot analysis of nuclear (N) and cytoplasmic (C) -enriched cell fractions of embryonal RMS cell lines. Nuclear EZH2 was detected in all cell lines. β-actin and topoisomerase IIβ were used as loading controls to discriminate the cytoplasmic and nuclear-enriched cell fractions, respectively. Representative of two independent experiments. (c) RD cells were transfected (Day 0) with EZH2 siRNA or control (CTR) siRNA and after 24 h transfected again (Day 1). Cells cultured in proliferating growth medium (GM, i.e. supplemented with 10% of fetal calf serum) were harvested and counted starting from 24 h from the first siRNA trasfection at the indicated time points. *P < 0.05 (Student’s t-test). Results from three independent experiments are shown; Bars, Standard Deviation (SD). (d) Western blot showing levels of EZH2 24 h and 48 h post-transfection with CTR or EZH2 siRNA in RD cells. β-actin served as loading control. Representative of four independent experiments. (e) Western blot showing histone H3 trimethylation on Lys27 (H3K27me3) and on Lys4 (H3K4me3) status 72 h after EZH2 or CTR siRNA transfection. Histone H3 was the loading control. Representative of three independent experiments.