Fig. 5. EZH2 is required for NEPC cells.

a Expression profiles of PRC subunits in PAC and NEPC samples. P-value adjusted by Bonferroni method. **P < 0.01, ****P < 0.0001. (All values below 0 were close to 0 and the green color was therefore not distinguishable on the right bar). b qPCR analysis of EZH2 and PHF19 expression in our tumor specimen cohort including 40 PAC and eight NEPC samples. ****P < 0.0001. c qPCR analysis of expression of four PRC genes in control PC and NEPC cells. **P < 0.01, ****P < 0.0001. d Western blotting of EZH1 and EZH2 in control PC and NEPC cells. e ChIP enrichment analysis of EZH2-binding site on miR-708 in control PC cells and NEPC cells. f Effect of EZH2 knockdown and EZH2 inhibition by GSK-126 on NEPC markers expression. EZH2-knockdown PC cells were treated with NEPC-inducing medium for 6 days, or control cells were treated with NEPC-inducing medium with GSK126 (2.5 μM) for 6 days. NEPC phenotype was evaluated by qPCR of NEPC markers CgA and SCG3. g Effects of EZH2 inhibitor GSK126, docetaxel (DTX), PI3K/AKT pathway inhibitor LY294002 alone, combination of GSK126 with DTX or LY294002, or DTX + LY294002 on cell viability of NEPC cells derived from C4–2. NEPC cells were treated with vehicle, GSK126 (5 μM), DTX (10 nM), LY294002 (LY, 5 μM), DTX + LY294002, DTX + GSK126, or DTX + GSK126 + LY for 48 h, and the percentages of viable cells were counted and calculated. *P < 0.05, **P < 0.01. h miR-708 expression in NEPC cells derived from C4–2 treated with the indicated drugs for 24 h. **P < 0.01, ****P < 0.0001. i Effects of anti-miR-708 on NEPC markers expression when EZH2 was inhibited. C4–2 cells were transfected with anti-miR-708 and then treated with NEPC-inducing medium for 6 days with or without GSK126. **P < 0.01. Error bars represent the standard deviation of biological triplicates