Fig. 7. ADT induces NE by downregulating REST through CREB1-activated EZH2 epigenetic repression.

A Western blotting on PC3 cells shows that REST is downregulated by CREB1 signaling activator forskolin (10 µM, left) or by overexpressing activated CREB1 cDNA (right), which is reversed by EZH2 inhibitor GSK126 (10 µM). B Western blotting shows that, when EZH2 is silenced by shEZH2, forskolin could no longer repress REST and induce NE marker ENO2 and H3K27me3 in PC3 cells. ChIP-qPCR measuring H3K27me3 histone mark levels on REST promoter, after treating with indicated CREB1 signaling modulators: propranolol (PROP, 15 µM) for 24 h in NE1.3 (C) and PC3 (D). ChIP-qPCR measuring EZH2 protein binding on REST promoter upon treating with these CREB1 signaling modulators in PC3 (E) and NE1.3 (F). G C4-2 cells were grown in FBS or CSS media for 7 days, then treated with DMSO or 10 µM MDV3100 for 3 days, followed by treatments of DMSO or 5 µM EZH2 inhibitor DZNeP for 2 days, and then Western blotting of REST and loading control GAPDH. H LNCaP cells were grown in FBS or CSS media for 5 days, then treated with DMSO or 10 µM EZH2 inhibitor EZP-6438 for 2 days. Whole cell lysates were collected and analyzed by western blotting for REST and loading control GAPDH. I C4-2 and 22Rv1 cells were grown in FBS or CSS media for 7 days and then EZH2 protein binding on the REST promoter region were measured by EZH2 ChIP-qPCR. The quantifications and graphs for these ChIP-PCRs are in Supplementary Fig. S3D.