FIGURE 4.

H₂S suppresses AR transactivation. A, PSA but not AR expression was up-regulated in LNCaP-B cells. *, p < 0.05 when compared with all groups. B, CSE knockdown induced PSA expression. After LNCaP cells were transfected with CSE-siRNAs or negative siRNA (Neg-siRNA) for 48 h, the cells were collected for Western blotting analysis. *, p < 0.05. C, H₂S inhibited PSA protein expression. Twenty-four hours after LNCaP cells were treated with 10 nm R1881 in the presence or absence of NaHS (30 μm), the cells were collected for Western blotting analysis of AR and PSA expression. D, H₂S inhibited the mRNA expression of PSA and TMPRSS2. Twenty-four hours after LNCaP cells were treated with 10 nm R1881 in the presence or absence of NaHS (30 μm), the cells were collected for real-time PCR analysis of AR and PSA mRNA expression. *, p < 0.05 versus R1881 group and NaHS group; #, p < 0.05 versus R1881 group. E, GYY4137 inhibited the expression of AR-targeted genes. Forty-eight hours after LNCaP cells were incubated with GYY4137 (100 μm), the cells were collected for analyzing the mRNA expression of PSA and TMPRSS2. *, p < 0.05. F, H₂S inhibited basal and R1881-induced PSA secretion in LNCaP cells. Forty-eight hours after LNCaP cells were incubated with R1881 (10 nm) with or without NaHS (30 μm), the medium was collected for PSA measurement. *, p < 0.05 versus R1881 group and NaHS group; #, p < 0.05 versus R1881 group. G, H₂S inhibited ARE luciferase activity. Forty-eight hours after LNCaP cells were transfected with inducible ARE luciferase construct in the presence or absence of R1881 (10 nm) and NaHS (30 μm), the cells were collected for measuring luciferase activity. *, p < 0.05 versus R1881 group and NaHS group; #, p < 0.05 versus R1881 group. H, CSE overexpression suppressed ARE luciferase activity. Twenty-four hours after LNCaP cells were transfected with Ad-CSE, the cells were transfected with inducible ARE luciferase construct in the presence of R1881 (10 nm) for another 48 h. *, p < 0.05 versus all other groups. I, H₂S inhibited AR binding with ARE in AR-targeted genes. Forty-eight hours after LNCaP cells were treated with R1881 (10 nm) with or without NaHS (30 μm), the cells were subjected to ChIP assay to detect the binding of AR with ARE-containing promoter region in both PSA and TMPRSS2. The typical binding images are shown in the left panel, and quantitative analysis of AR and the promoter interaction measured by real-time PCR is shown in the right panel. *, p < 0.05 versus R1881 group and NaHS group; #, p < 0.05 versus R1881 group. All experiments were repeated at least three times. Data are presented as means ± S.E.