fig20

Supplementary Figure 2. TGF-β induces EMT phenotypes in BT474 cells. (A) BT474 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) BT474 cells were stimulated with TGF-β1 (5 ng/mL) for 0-96 h as indicated, at which point differences in the expression of E-cad and N-cad mRNA (top) and protein (bottom) were determined. mRNA data are the mean foldchanges (± SE; n = 3; *P < 0.05; Student's t-test), while protein data are representative images from 3-independent experiments. (C and D) BT474 cells were treated with TGF-β1 (5 ng/mL) for 0-96 h, at which point the expression of b-catenin, CK19, and ZO-1 (C) or of MMP-9, Twist-1, and vimentin (D) were monitored by real-time PCR. Data are the mean fold-changes (± SE; n = 3;*P < 0.05; Student's t-test). (E and F) BT474 cells were stimulated with TGF-β1 (5 ng/mL) for 72 h, and subsequently were fixed in paraformaldehyde and processed for indirect immunofluorescence of either E-cad (E) or b-catenin (F), as well as for direct immunofluorescence of actin (E and F). Nuclei were visualized by DAPI staining. Images are representative from 3-independent experiments (× 400). TGF: transforming growth factor; EMT: epithelial-mesenchymal transition; SE: standard error