Fig. 6. EZH2 represses TBX3 in RD cells and promotes differentiation of ARMS and ERMS cells.

a RH30 shEZH2 cell lines were assayed by immunofluorescence with antibodies against MyHC after 4 days of differentiation. DAPI was used to stain the nuclei. Images were taken at ×100 and scale bars represent 100 μm. b–d The mRNA expression of TNNI2 (b), MYOD1 (c), and MYOG (d) were assayed in RH30 shEZH2 cell lines after 4 days of differentiation (D4) by qRT-PCR. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus scr. e Protein extracts from RH30 cells as in (b) were used for western blot analysis with antibodies against the indicated proteins. GAPDH was used as the loading control. f EZH2 represses TBX3 in RD cells. RD cells were transiently transfected with shEZH2 and scr control and assayed by mRNA expression of the indicated genes by qRT-PCR. Error bars, S.E. and ***p ≤ 0.001 versus scr. g RD cells were transiently transfected with shEZH2 and scr control and assayed by immunofluorescence with antibodies against MyHC after 2 days of differentiation (D2). DAPI was used to stain nuclei. Images were taken at ×100 and scale bars represent 100 μm. Nuclei per myofiber are quantitated in the right panel. Error bars, S.E., and n.s. represent not statistically significant versus scr. h The mRNA expression of EZH2 in the cells shown in (g) was assayed by qRT-PCR and shown in the right panel. Error bars, S.E. and ***p ≤ 0.001 versus scr