Fig. 7: EGR1 is correlated with differentiation and repressed by EZH2 in RH30 cells.
From: TBX3 represses TBX2 under the control of the PRC2 complex in skeletal muscle and rhabdomyosarcoma
a, b mRNA expression of EGR1 (a) and NDRG1 (b) was assayed by qRT-PCR in RH30 shEZH2 and scr cell lines. Error bars, S.E. and ***p ≤ 0.001 versus scr. c TBX3 does not induce EGR1. mRNA expression of EGR1 was assayed by qRT-PCR. Error bars, S.E. and *p ≤ 0.05, **p ≤ 0.01 versus scr. n.s. represents not statistically significant. d, e Inhibition of EZH2 induces EGR1. RH30 cells were treated with the indicated concentration of GSK126 or DMSO (vehicle control) for 72 h and harvested for RNA (d) and protein (e). Error bars, S.E. and *p ≤ 0.05, ***p ≤ 0.001 versus DMSO. f EZH2 binds the EGR1 promoter. ChIP assays were performed on RH30 cells using antibodies against EZH2 and primers against the EGR1 promoter. Error bars, S.E. and ***p ≤ 0.001 versus IgG. g The EGR1 promoter contains H3K27me3. ChIP assays were performed on RH30 cells using antibodies against H3K27me3 and primers against the EGR1 promoter. Error bars, S.E. and ***p ≤ 0.001 versus IgG. h Stable RD cell lines overexpressing TBX3 were transiently transfected with shEGR1 or scr when confluent and assayed by immunofluorescence for MyHC after 2 days of differentiation conditions (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 **p ≤ 0.01 versus scr. i Cells as in (h) were analyzed for protein expression with the indicated antibodies and replicate blots are quantitated in the right panel. TUBULIN was used as the loading control. Error bars, S.E. and **p ≤ 0.01 versus scr. n.s. represents not statistically significant
