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. 2008 Oct 23;150(3):1259–1268. doi: 10.1210/en.2008-0858

Figure 1.

Figure 1

Testosterone induced myogenic differentiation and promoted nuclear translocation of β-catenin and its colocalization with AR in C3H 10T1/2 cells. A, Top, C3H 10T1/2 cells, pretreated with 5′-azacytidine, as described in Materials and Methods, were treated with medium alone (Con) or 100 nm testosterone (T) for 3 d (for MyoD), 6 d (for myogenin), or 12 d (for MHC II), and Western blot analysis was performed using 30 μg total cell lysate using anti-MyoD, myogenin, or MHC II antibodies. One representative gel has been shown of three independent experiments. Bottom, Quantitative densitometric analysis of Western blots from three independent gels after normalization with GAPDH. Data are mean ± sem. Student’s t test was performed for statistical analysis (*, P ≤ 0.05; **, P ≤ 0.01). B, Double-immunofluorescence analysis was performed to detect AR (green) and β-catenin (red). Colocalization of AR and β-catenin is indicated by yellow immunofluorescence in the merged fields. DAPI (blue) counterstain was used to localize the nuclei. Three independent experiments were performed, and a representative photograph is shown. C, Top, Cells were treated with or without testosterone (100 nm) for various time points as indicated. Three independent experiments were performed, and a representative gel is shown. Bottom, DHT-induced (10 nm DHT) nuclear translocation of β-catenin was restored to control levels by coincubation with AR antagonist bicalutamide (Bic). The experiment was repeated twice, and a representative gel is shown. Bic, Bicalutamide; Con, control wells; Cyto, cytoplasmic; Nuc, nuclear. D, Physical interaction of AR, β-catenin, and TCF-4 proteins in DHT-treated (0–30 nm) C3H 10T1/2 cells. Cells were also coincubated with 300 nm bicalutamide (Bic) along with DHT (10 nm) in some cases. The immunoprecipitation was performed using either anti-AR antibody or a nonspecific IgG, and the immunoprecipitates were analyzed by Western blot analysis using anti-AR, anti-β-catenin, or anti-TCF-4 antibodies. Three independent experiments were performed, and data from a representative experiment are presented.