Fig. 8. Upregulation of β-catenin after Wt1 deletion in Sertoli cells. (A) Nuclear localization of β-catenin in Sertoli cells of Wt1^fx/-; AMH-Cre^tg/+ mice at E15.5. β-Catenin protein was stained by an anti-β-catenin antibody (green) and nuclei were counterstained with DAPI (blue). β-Catenin was normally located on Sertoli cell membrane in control testes (left panel, arrowheads). However, β-catenin was detected in Sertoli cell nuclei in testes of Wt1 conditional knockout mice (right panel, arrows). Scale bar: 20 µm. (B) Immunofluorescent images of isolated primary Sertoli cells stained with WT1 (green). Nuclei were counterstained with DAPI (blue). Over 90% of the cells were WT1 positive. (C) Real-time PCR quantification of Wt1 expression in isolated primary Sertoli cells of mutant and control mice, with or without 4OH-tamoxifen treatment. Numerical data present mean±s.e.m. of relative expression of Wt1 in three independent experiments. Column 1, Wt1^fx/fx; +/+ with treatment; column 2, Wt1^fx/-; CAGGCre-ER^tg/+ without treatment; column 3, Wt1^fx/-; CAGGCre-ER^tg/+ with treatment. Wt1 expression was significantly reduced upon 4OH-tamoxifen treatment in Wt1^fx/-; CAGGCre-ER^tg/+ Sertoli cells. (D) Western blot to analyze the expression levels of total β-catenin and the active form of β-catenin in isolated primary Sertoli cells of mutant and control mice, with or without 4OH-tamoxifen treatment. The levels of total β-catenin and the active form of β-catenin were upregulated upon 4OH-tamoxifen treatment in Wt1^fx/-; CAGGCre-ER^tg/+ Sertoli cells (quantification on the right).