Figure 6.

The essential roles of PIAS1 for Smad4-mediated p21^WAF1/Cip1 promoter binding, cell apoptosis and inhibition of proliferation. (a) LNCaP cells were transfected with PIAS1-shRNAs for immunoblot analysis with PIAS1 antibody. (b) The increased binding of Smad4 to SBE1 and SBE3 regions in the p21^WAF1/Cip1 promoter was reversible by PIAS1-shRNA. LNCaP cells transfected with PIAS1-shRNA1 or the control vector were treated with 150 μM ZnSO₄ for 24 h. Cross-linked chromatin complexes were immunoprecipitated with anti-Smad4 or IgG antibody. Co-precipitated DNA sequences were amplified using specific primers spanning the SBE1 and SBE3 in the p21^WAF1/Cip1 promoter. (c) LNCaP cells were transiently cotransfected with plasmids as indicated, and then treated with 125 μM ZnSO4 for 24 h. The apoptotic percentages of cells were measured by flow cytometry. Columns, mean (n=3); bars, S.D. **P<0.01. ***P<0.001 (Student's t-test). (d) LNCaP cells were transfected with Smad4-shRNA, Smad2-shRNA, or PIAS1-shRNA alone or together, and then cultured in the T medium containing 150 μM ZnSO₄ for 12 days. Colonies containing more than 50 cells were counted. Columns, mean (n=3); bars, S.D. *P<0.05. **P<0.01. ***P<0.001. All versus mock-transfected zinc-treated control group (Student's t-test)