Trichostatin A (TSA) reduced growth and altered differentiation and self-renewal capacity of ERMS cell lines.

(A) Western blots demonstrating hyperacetylation of histones using antibodies against acetyl-histone H3 (Lys9), acetyl-histone H3 (Lys27), acetyl-histone H4 (Lys5), and acetyl-histone H4 (Lys8). D: DMSO; S: vorinostat (SAHA); T: TSA. Each band intensity was normalized to GAPDH loading control. Relative fold increase to DMSO (vehicle) treatment is shown. (B) Analysis of cell viability by cell counts. Cell counts were performed on cells treated with DMSO, 200 nM TSA or 1 μM SAHA at day 0 and day 5. Fold change in cell counts was normalized to day 0. (C-D) Representative images of immunofluorescence (IF) against MF20 performed on RD cells treated with DMSO (C) or 200 nM TSA (D) for 3 days in 2% horse serum in DMEM. Green: MF20-positive cells. Blue: DAPI. Scale bar: 20 μm. (E) Summary of IF against MF20 in ERMS (RD, 381T and SMS-CTR) and ARMS (Rh3, Rh5 and Rh30) cell lines treated with DMSO, 200 nM TSA or 1 μM SAHA. (F) Chromatin immunoprecipitation (ChIP) assays showing differential binding of acetyl-histone H3 (Lys9) at myogenic promoters. Fold enrichment binding of MYOD1, MYOG, and MYH4 promoter regions were determined by quantitative PCR, normalizing amplification levels to input DNA of each sample. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (G-H) Representative bright-field images from a sphere assay on RD cells treated with DMSO (G) and 200 nM TSA (H). (I) Summary of sphere assays in RD and 381T cells. Each error bar in panels (B), (E), (F) and (I) indicates standard deviation of 3 technical replicates. * indicates p