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Supplementary Figure 1. TGF-β induces EMT phenotypes in MCF-7 cells in both 2D- and 3D cultures. (A-C) MCF-7 cells were propagated in either 2D- or 3D-culture systems and treated with TGF-b1 (5 ng/mL) for either 72 h (A and B) or 96 h (C) in 2D-cultures, or for either 8 days (A and B) or 96 h (C) as indicated. Afterward, total RNA has harvested and subjected to real-time PCR analyses to monitor the expression of the epithelial markers E-cad and CK19 (A), of the mesenchymal markers vimentin and N-cad (B), or of Cox-2 (C). (D) MCF-7 cells were stimulated with TGF-b1 for 0-96 h as indicated, at which point the levels of MMP-9 were monitored by realtime PCR. Data are the mean fold-changes (± SE; n = 3; *P < 0.05; Student's t-test). (E) MCF-7 cells were stimulated with TGF-b1 (5 ng/mL) for 96 h, and subsequently were fixed in paraformaldehyde and processed for indirect immunofluorescence of b-catenin, as well as for direct immunofluorescence of actin. Images are representative of 2-independent experiments (×400). TGF: transforming growth factor; EMT: epithelial-mesenchymal transition; SE: standard error