Fig. 5

TSP1 is a novel EZH2 epigenetic target in prostate cancer cells. a, b Changes in TSP1 expression and H3K27me3 levels upon EZH2 cDNA overexpression (a) and shRNA silencing (b) in prostate cancer cells. c Marked reduction of H3K27me3 and up-regulation of TSP1 in PC3, RWPE, and NE1.3 cells treated with 5 μM of EZH2 inhibitor DZNEP or GSK126 for 24 h. d PC3 cells were treated with 2.5 μM DZNEP or vehicle control (DMSO) for 24 h. A ChIP assay was conducted using IgG control or anti-H3K27me3 antibody. The level of H3K27me3 mark on TSP1 promoter was presented as % of the inputs (Y-axis), which was confirmed through DNA gel electrophoresis. e PC3 cells were transfected with pGL3 firefly luciferase vector with TSP1 promoter along with a pRL-TK renilla luciferase control vector, then treated with DZNEP 2.5 μM and 5 μM for 24 h, followed by Dual-luciferase reporter assay. Plotted on Y-axis are the ratios of firefly luciferase signals vs renilla luciferase control signals in each treatment. f PC3 cells carrying an empty vector or siRNA-resistant EZH2 cDNA were transfected with control siRNA (siCNL) or siEZH2. Total RNA was extracted 72 h after siRNA treatment, and RT-qPCR for EZH2 and TSP1 was carried out, using GAPDH as the internal control. Plotted on Y-axis are average fold changes with s.d.