Aberrant NOTCH1-mediated signaling activity is essential for inhibiting myogenic differentiation of ERMS.

(A) Summary of MF20 IF of RD cells treated with two different doses of GSI-IX, PD173074, or cyclopamine. (B-C) Representative MF20 IF images of RD cells treated with DMSO (B) and 20 μM GSI-IX (C). Scale bar = 20 μm. (D) Western blot of NOTCH1 expression in RD cells treated with DMSO, 200 nM TSA, or 1 μM SAHA. Each band intensity was normalized to GAPDH loading control. Percent intensity relative to DMSO (vehicle) treatment is shown. (E) Quantitative RT-PCR analysis of NOTCH1 pathway downstream targets, HEY1, HEY2 and HES1 in RD cells treated with DMSO, 200 nM TSA, or 1 μM SAHA. (F) ChIP assay summary showing differential binding of acetyl-histone H3 (Lys9) at the NOTCH1 promoter in RD cells treated with DMSO or 200 nM TSA. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (G) Summary of MF20 IF of control GFP-overexpressing and NICD-overexpressing RD cells treated with DMSO, 200 nM TSA or 1 μM SAHA. Error bar in each graph indicates standard deviation of technical triplicates. Brackets in each group are used to indicate comparison groups. * indicates p