Figure 5

CS-99 human carcinosarcoma cells undergo MET in vitro. (a) Cultured CS-99 cells possess cell–cell contacts, interspersed with spindle-shaped cells. CS-99 cells transfected with Rint express DsRed, while cells transfected with RIIIcI² express only background levels of DsRed. (b) CS-99 cells express FGFR2-IIIc mRNA. Epithelial DT and mesenchymal AT3 cells are included as controls for the IIIb and IIIc isoforms, respectively. For each cell type, RT–PCR products are mock digested (M), digested with the FGFR2 IIIb-specific enzyme, AvaI (A), or digested with the FGFR2-IIIc-specific enzyme, EcoRV (E). (c) CS-99 cells lack E-cadherin and express vimentin. Epithelial LNCaP and mesenchymal PC3 cells are included as controls. (d) Reverse transfection of miR200a, miR200b and miR200c into CS-99 cells leads to a morphological change consistent with MET; cells change shape from spindle-like, single cells to clusters of rounded cells. (e) Expression of miR200s in CS-99s induces upregulation of E-cadherin and loss of vimentin. (f). The miR200 targets, ZEB1 and Slug, are downregulated and upregulated, respectively. (g) Induction of MET in CS-99 cells has no effect on luciferase expression from a control reporter, FFint, but a significant increase in luciferase expression is observed from the E-cadFFIIIcI² MET reporter.