FIG. 6.

Snail mediates control of RIN1 expression by TGF-β. (A) Cells and conditions as in Fig. 5A were used, and levels of endogenous SNAI1 (Snail) mRNA were determined by quantitative real-time PCR. (B) A mouse Rin1 promoter-luciferase construct (pRPluc) was transfected into HeLa cells together with vector (ctr), SNAI1 (SNAI), or a repressor domain deletion mutant of SNAI1 (SNAI^m). Luciferase activity was measured and normalized to Renilla luciferase activity and plotted as means ± SDs of triplicates from a representative of at least two independent experiments. (C) Normalized real-time PCR measurements of SNAI1 and RIN1 mRNAs following treatment of MDA-MB-231 cells with SNAI1-directed or control siRNA. (D) MDA-MB-231 cells were treated with SB216763 (GSK3β inhibitor) and MG-132 (proteosome inhibitor) for 6 h or left untreated (ctr). Immunoblots of endogenous SNAI1, RIN1, CDH1, and TUBB are shown (CDH1 was immunoprecipitated prior to immunoblotting). (E) EGF (100 ng/ml, 18 h) stimulation of MDA-MB-231 cells in the absence (−) or presence (+) of wortmannin (Wort). Levels of SNAI1, phospho-AKT, and total AKT protein were measured by immunoblotting (left). Normalized levels of RIN1 mRNA were measured by real-time PCR (right).