Figure 3.

TGF-β treatment has no effect on the expression of AR and AR targets. (A) TGF-β did not induce phospho-SMAD3 or phospho-SMAD2 in Rv1 or C4-2 cells. Indicated cells were treated with 10 ng/ml of TGF-β for 1 h. Western blot analysis was performed using the indicated antibodies. (B and C) Real-time RT-PCR results showing that TGF-β treatment (10 ng/ml for 6 h) of Rv1 (B) or C4-2 (C) cells had no effect on transcript levels of AR, AR-V7 and AR targets. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (ns, not significant). (D) Real-time RT-PCR results showing the decreased transcript levels of AR, AR-V7 and AR targets in the SMAD3-KD VCaP cells. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (***P < 0.001). (E) Western blots showing the decreased level of AR and AR-V7 in the SMAD3-KD VCaP cells. (F) Western blots showing the increased levels of phospho-SMAD3 and phospho-SMAD2 in VCaP cells after TGF-β treatment (10 ng/ml for 1 h). (G) Real-time RT-PCR analysis of VCaP cells showing no effect of TGF-β treatment (10 ng/ml for 6 h) on transcript levels of AR, AR-V7 and AR targets relative to untreated cells. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (ns, not significant; *P < 0.05). (H) Decreased levels of phospho-SMAD3 after treating PC3 cells with SIS3 for 24 h. (I) No changes of AR or AR-V7 levels after treating Rv1 cells with SIS3 for 24 h. (J) Effect of SIS3 treatment on transcript levels of AR, AR-V7 and AR target genes. Rv1 cells were treated with indicated concentration of SIS3 for 24 h before real-time RT-PCR analysis. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; ***P < 0.001).