Fig. 8

ADT and CREB activation downregulate TSP1 through EZH2-mediated epigenetic repression. a Western blotting for TSP1 and p-CREB levels in 22Rv1 cells treated with 10 μM MDV3100 for 72 h without or with 5 nM of DHT for 24 h. b LNCaP cells were grown in CSS medium for 48 h, without or with subsequent treatment of 10 μM PKA inhibitor PKI for 5 h. TSP1 and p-CREB protein levels were assessed by western blotting. c Protein level changes of p-CREB, TSP1, and NE markers in LNCaP-Dox-ACREB cells treated by MDV3100 with and without Dox for 72 h. ACREB, a CREB inhibitory polypeptide, is induced by Dox. d TSP1 expression in PC3 cells overexpressing empty vector, wild-type (WT) or constitutively active mutant (Y134F) of CREB cDNA. e PC3 cells expressing empty vector or CREB-Y134F were infected with pLKO.1 lentivirus for shEZH2, followed by the examination of TSP1 expression. f PC3 cells were treated with 10 μM PKA/CREB activator forskolin (FSK) with or without EZH2 inhibitor GSK126 (5 μM) for 24 h. Levels of indicated proteins were measured by western blotting. g PC3 cells expressing shEZH2 were treated with 10 μM PKA/CREB activator ISO for 24 h. PC3 cells expressing Scramble control shRNA were used as control. h LNCaP and PC3 cells were treated with 10 μM PKA/CREB signaling activator ISO, with or without 10 μM PKA/CREB signaling inhibitor PRO, for 24 h, followed by ChIP assay using anti-H3K27me3 or IgG control antibody. The amount of H3K27me3 histone marks on TSP1 promoter was measured by quantitative PCR and presented as % of the input (Y-axis). DNA gel electrophoresis was performed to visualize the PCR results. i RT-PCR for TSP1mRNA changes in PC3 and LNCaP cells that were treated similarly as in the ChIP-qPCR above. j LNCaP cells were treated either with DMSO, 10 μM MDV3100 without or with 5 μM GSK126 for 72 h. RT-PCR was done for TSP1 and beta actin