Fig. 3

EZH2, but not the PRC2 complex, is essential for Enz treatment induced NED. a, b Enz-increased NE markers were reduced after EZH2 knockdown. The C4-2 pLKO control and shEZH2 cells were treated with/without (w/o) 10 μM Enz for 4 days, and the NE markers detected by (a) western blot (WB) and (b) qPCR. c EZH2 knockdown can reverse the Enz-induced NED. The C4-2 pLKO and shEZH2 cells were treated w/o Enz for 7 days, then the cell morphology was observed under microscope, quantitations in lower panel. (Scale bar = 20 μm). d EZH2 inhibitors can inhibit the expression of NE markers. C4-2 cells were treated w/o Enz and EZH2 inhibitors (4 μM GSK126 and 1 μM Dznep) for 4 days, and the mRNAs of NE markers analyzed by qPCR. e Expression levels of NE markers in NE1.8 cells after knocking down EZH2. f C4-2 pWPI and pWPI-lncRNA-p21 cells were treated w/o 4 μM GSK126, and the NE markers levels detected by qPCR. g H3K27me3 levels in C4-2 cells after treating w/o Enz for 4 days. h H3K27me3 levels in C4-2 cells after DMSO, Enz and GSK126 treatment. i Co-IP assay to detect the interaction between EZH2, SUZ12 and EED in C4-2 cells w/o Enz treatment for 4 days. j GSEA of PRC2 and EZH2 pathway enrichment in C4-2 cells after Enz treatment. k Expression of PRC2 complex target genes in C4-2 cells after 4 days of Enz treatment. l The mRNA levels of NE markers in C4-2 pLKO control and C4-2 shEED cells after Enz treatment for 4 days. For b, d, f, k, the data are presented as mean ± SD, *p < 0.05, **p < 0.005, by t-test