FIGURE 4 from Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer

BET inhibitors suppress ALDH1A3 expression and growth of DTP cells. A, Effects of BET inhibitors on 5-FU–induced mRNA expression of ALDH1A3 in JSC15-3 (left) and JSC18-1 (right) cells. Cells were treated with 3 µmol/L 5-FU in the presence or absence of 1 µmol/L OTX015 or 3 µmol/L I-BET-762 for 5 days. Then, RNAs were prepared and subjected to qRT-PCR. B, FACS analysis of ALDH1A3-GFP (left) and ALDH1A3low-GFP (right) JSC15–3 cells. Cells were treated as described in A and subjected to flow cytometry. In A and B, experiments were conducted with three technical replicates and were repeated at least three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA. Effects of BET inhibitors on parental and 5FU-TP JSC15-3 (C) and JSC18-1 (D) cell growth. The 5FU-TP cells were prepared by treating the respective parental cells with 3 µmol/L (JSC15-3) or 1 µmol/L (JSC18-1) 5-FU for 6 days. Then, the cells were reseeded and treated with the indicated concentrations of OTX015 (left) and I-BET-762 (right) for 6 days. Experiments were conducted with six technical replicates and were repeated at least three times. *, P < 0.05; **, P < 0.01, one-tailed Mann–Whitney rank-sum test. E, Effects of BET inhibitors on expression of exogenous ALDH1A3 in JSC15-3 cells. Mock and ALDH1A3-O/E JSC15-3 cells were treated as described in A and subjected to qRT-PCR analysis. Experiments were conducted with three technical replicates and were repeated at least three times. F, Effects of BET inhibitors on mock and O/E-derived DTP cell growth. Cells were treated as described in C, and cell numbers were quantitated. Experiments were conducted with six technical replicates and were repeated three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA. ns, not significant.