PRC2 regulates ICC-SC aging.(A) Activation of TRP53 by nutlin 3a (30 μmol/L, 72 hours) upregulated EZH2 protein and increased H3K27me3 levels in D2211B ICC-SC without affecting TRP53 phosphorylation (P-TRP53). The 150-fold less potent enantiomer nutlin 3b (30 μmol/L, 72 hours) used as control for nutlin 3a. (B–E) Ezh2 inhibitors (EPZ6438 [EPZ] and GSK126 [GSK]) applied at 500 nmol/L for 72 hours prevented nutlin 3a (30 μmol/L, 72 hours)-induced H3K27me3 (n = 8–9/group) and cell growth arrest (n = 15/group) without affecting TRP53 and EZH2 protein expression in ICC-SC (D2211B cells). The inactive enantiomer nutlin 3b (3b; 30 μmol/L) served as a control for nutlin 3a. (F and G) siRNA-mediated knockdown Ezh2 (siEzh2) prevented nutlin 3a-induced EZH2 upregulation (n = 9/group) and cell growth arrest (n = 30/group) without affecting TRP53 protein expression and H3K27me3 levels in ICC-SC (D2211B cells). Corresponding Scr served as a control for siEzh2. Statistical significance was determined using Kruskal-Wallis 1-way analysis of variance (on ranks). ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001. ns, not significant.