fig1

Figure 1. TGF-β induces EMT in MCF-7 cells and potentiates noncanonical TGF-β signaling. (A) MCF-7 cells were treated with TGF-β1 (5 ng/mL) for 0-72 h to induce an EMT program. Photomicrographs depict accompanying alterations in cell morphology (×400); (B and C) MCF-7 cells were stimulated with TGF-β1 (5 ng/mL) for 0-96 h, at which point total RNA was harvested and subjected to real-time PCR to monitor differences in the expression of Snail and Twist (B), and of Zeb1 and Zeb2 (C). Data are the mean fold-changes (± SE; n = 3; *P < 0.05; Student's t-test); (D) MCF-7 cells were treated with TGF-β1 (5 ng/mL) for 0-96 h, at which point detergent-solubilized extracts were immunoblotted for phospho-Tyr (pY), β-catenin (β-cat), or β-actin as indicated (top). Additionally, E-cad immunocomplexes were captured and immunoblotted for β-catenin (β-cat) and E-cad as indicated (bottom). Images are representative of 3-independent experiments. (E-G) Pre- and post-EMT MCF-7 cells were transiently transfected overnight with the pSBE-(E), TopFlash-(F), or p3TP-(G) luciferase reporter genes, as well as the pCMV-β-galactosidase reporter gene to control for differences in transfection efficiency. Afterward, the transfectants were stimulated overnight with TGF-β1 (5 ng/mL) prior to measuring luciferase and β-gal activities. Data are the mean fold-changes (± SE; n = 3;*P < 0.05; Student's t-test). TGF: transforming growth factor; EMT: epithelial-mesenchymal transition; SE: standard error