Figure 2.

Dual inhibition of EZH1 and EZH2 in MRT cells

(A) The effect of EZH1/2 dual inhibitors and EZH2-selective inhibitors on the cell growth. Six MRTs (A204.1, TTC642, G401.6TG, KYM-1, JMU-RTK-2, and TTC549) and SMARCB1 wild-type RMS (RD) cells were treated with DS-3201b, UNC1999, CPI-360, GSK126, or EPZ-6438 for the indicated durations, and relative cell growth was evaluated by a WST-8 assay (n = 3, means ± SD). Differences were statistically evaluated by two-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance between control and each drug treatment (DS-3201b, UNC1999, CPI-360, GSK126, or EPZ-6438; 100 nM) is p < 0.001 in all MRT cell lines on day 14. (B) Relative expression levels of EZH1 and EZH2 mRNAs in steady state of each cell line (n = 3, means ± SD). (C) The methylation levels of H3K27 after treatment with EZH1/2 dual inhibitors and EZH2-selective inhibitors. Cells were treated with DS-3201b, UNC1999, CPI-360, GSK126, or EPZ-6438 (100 nM each) for 7 days, followed by western blotting analysis. The numbers below H3K27me3 indicate each band density relative to control (taken as “1”). (D and E) Expression of EZH1 and EZH2 proteins (D) and mRNAs (E) in DS-3201b- or EPZ-6438-treated cells. Cells were treated with DS-3201b (100 nM) or EPZ-6438 (100 nM) for 7 days, followed by western blotting and qRT-PCR, respectively. (D) The numbers below EZH1 indicate each band density relative to control (taken as “1”). (E) n = 3, means ± SD. The expression level of the control is indicated as “1.” ∗∗∗p < 0.001 versus control.