Fig. 2

ING3 regulates the AR pathway. a LNCaP (left panel) or C4-2 (right panel) cells transfected with GFP or ING3 constructs were grown for 24 h +/– 10 nM MB and were tested for PSA, TMPRSS2, and FKBP5 expression by qRT-PCR (ANOVA **P < 0.01, ***P < 0.001). b, c LNCaP cells transfected with siCtrl or siING3 for 24 h were treated with 10 nM MB for 24 h. Levels of ING3 (b) or androgen-regulated genes (c) were assessed by qRT-PCR (ANOVA ***P < 0.001). d LNCaP cells transfected with empty vector or ING3-HA were harvested 24 h later, and lysates were blotted with α-HA, α-PSA, or α-actin. Lysates of C4-2 cells transfected with siCtrl or siING3 for 48 h were blotted for the proteins indicated. e C4-2 cells transfected with siCtrl or siING3 for 24 h were untreated or treated with 10 nM MB for 24 h. ChIP assays using α-AR antibody and AR-bound DNA used primers specific for an ARE on the FKBP5 gene (t test *P < 0.05). f HEK293T cells were co-transfected with 1 μg AR3-tkk-LUC, 0.2 μg β-gal, 0.1 μg GFP, and 20 nM of either siCtrl or siING3 for 48 h +/– MB for the final 24 h. LUC reporter activity was normalized to β-gal (t test **P < 0.01). g Cells were co-transfected with 1 μg AR3-tkk-LUC, 0.2 μg β-gal, 1 μg AR, and either empty vector or the indicated amounts of ING3 expression plasmid for 48 h +/– MB in the indicated amounts (ANOVA ***P < 0.001). h Cells were co-transfected with 1 μg AR3-tkk-LUC, 0.2 μg β-gal, 1 μg AR, and 0.2 μg of either empty vector (Ctrl) or full-length ING3 expression plasmid. 1 nM MB was added for the indicated time points (t test *P < 0.05, ***P < 0.001). i The map of the plant homeodomain (PHD) deleted construct is shown. Cells were co-transfected with 1 μg AR3-tkk-LUC, 0.2 μg β-gal, 1 μg AR, and either empty vector, full-length ING3 expression plasmid, or PHD deletion mutant in CSS medium for 48 h. Levels of ING3-HA expression were verified by western blotting (left), and ARE-driven reporter expression is shown in response to full-length and PHD-deleted ING3 (right, t test *P < 0.05). j Co-precipitation using HEK293T cells co-transfected with full-length AR, ING3, and an ING3 deletion mutant. HA beads were used to immunoprecipitate ING3 constructs. The interacting AR and endogenous TIP60 were detected by western blotting with their respective antibodies