Figure 5.

Testosterone up-regulates Smad 7 and Fst protein expression both in vitro and in vivo. A, Top, Control or TGF-β1-treated (5 ng/ml) C3H 10T1/2 cells were incubated with or without testosterone (T) for 6 h, and 40 μg total protein lysates were analyzed by Western blot analysis using anti-Smad7 or anti-GAPDH antibody. The experiment was repeated three times, and a representative blot is shown. Bottom, Quantitative densitometric analysis of Western blots from three independent experiments after normalization with GAPDH is shown. Data are mean ± sem. *, P ≤ 0.05 vs. control (Con) group; #, P ≤ 0.05 vs. testosterone (T) group; ##, P ≤ 0.05 vs. TGF-β group. ANOVA was used to compare the difference between different groups, and individual groups were compared by using Tukey’s procedure. B, Immunocytochemical staining of C3H 10T1/2 cells for Smad7 expression. Control or TGF-β1-treated (5 ng/ml) C3H 10T1/2 cells were incubated with or without testosterone (T) for 6 h in six-well chamber slides, fixed with 2% paraformaldehyde, and stained with anti-Smad7 antibody. C and D, Quantitative real-time RT-PCR analysis of Fst (C) and Smad7 (D) mRNA levels in levator ani muscle. Total RNA isolated from levator ani muscle of 6-wk-old C57BL/6J male mice were subjected to RT and quantitative real-time PCR using gene-specific primers. Sham indicates sham operated; Cast, castrated with blank SILASTIC brand implants; 1 cm, castrated with 1-cm testosterone implants; and 2 cm, castrated with 2-cm testosterone implants. Treatment duration was 14 d. RNA was isolated from levator ani muscle of different animals, and quantitative real-time PCR was performed three times. Quadruplicate samples were analyzed each time for each treatment group. Data are presented as mean ± sem. Statistical analysis was performed by using ANOVA; pairwise comparisons between groups were performed by using Tukey’s procedure.