Fig. 5. EZH2 is upregulated in RMS and targets TBX3.

a Expression of EZH2 was examined by western blot assays for RMS cell lines and proliferating C2C12 cells (UD) and after 6 days of differentiation (D6). Replicate blots are quantitated in the lower panel. GAPDH was used as a loading control. b–d RH30 cells were transfected with shEZH2 and scrambled control (scr) and two independent clones were characterized following selection for stable isolates. Expression of EZH2 (b), TBX3 (c), and TBX2 (d) were assayed by qRT-PCR. Error bars, S.E. and *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus scr. e Protein extracts from the cells in (b) were used for western blot assays with antibodies against the indicated proteins. GAPDH was used as a loading control. f Depletion of EZH2 impairs proliferation. RH30 cells as in (b) were seeded with the same number of cells and harvested for cell counts every 2 days. Error bars, S.E. and ***p ≤ 0.001 versus scr. g Scratch assays were performed for the RH30 cells as in (b). Images were taken at ×100 magnification. Migration rate was quantitated in the lower panel. Error bars, S.E. and *p ≤ 0.05, ***p ≤ 0.001 versus scr. h Soft agar assays were performed on the cells as in (b). Images were taken at ×100 magnification and the number of colonies quantified by counting in five random fields. Error bars, S.E. and ***p ≤ 0.001 versus scr