Fig. 7. Loss of TGFß-mediated ß-catenin degradation and TGFß responsiveness in Smad4^–/– cells. (A,B) Western blot showing ß-catenin in Smad4^+/+ (A) and Smad4^–/– (B) cells after they were treated with 2 ng/ml of TGFß for 24 hours. Samples 1 and 2 were mammary tumor cell lines derived from Brca1 conditional knockout and MMTV-ras transgenic mice, respectively. Sample 3 was a cell line derived from a P16 mammary gland from a Smad4^+/+ mouse. Sample 4 and 5 were two Smad4^–/– cell lines derived independently from mammary abscesses of Smad4^Co/CoWAP-Cre mice. Treatment of TGFß(+) led to a decrease in ß-catenin in all Smad4^+/+ cell lines, whereas no changes were detected in both independently derived Smad4^–/– cells. (C) Loss of Smad4 blocks TGFß responsiveness as Smad4^–/– cells did not undergo EMT and failed to decrease ß-catenin upon TGFß treatment. (D) Timecourse response of Smad4^+/+ (wild-type) cells to TGFß induced EMT and downregulation of ß-catenin. Scale bars: 100 µm for phase contract images and 39 µm for immunofluorescent images.