Fig. 6. In mice, TGF-ß1 is unable to inhibit ductal growth of Wnt5a^-/- epithelium in vivo and branching of Wnt5a^-/- organoids in vitro. (A) Carmine-stained BSA-treated and the contralateral TGF-ß-treated wild-type mammary gland confirms that the TGF-ß1 pellets (P) are functional and result in degeneration of the end bud and inhibition of ductal elongation. Insets represent magnified images of the end buds. (B) Carmine-stained glands reconstituted with Wnt5a^-/- epithelium. The left gland was treated with BSA and the right contralateral gland was treated with TGF-ß1. TGF-ß is unable to inhibit the end bud in the absence of Wnt5a. Insets represent magnified images of the end buds. (C) Ki67 staining in the end buds of wild-type mice either treated with BSA or TGF-ß. Treatment with TGF-ß resulted in reduced staining relative to BSA-treated glands. (D) Ki67 staining in the end buds of Wnt5a^-/- reconstituted glands treated with BSA or TGF-ß. Staining was similar in BSA- and TGF-ß-treated end buds. (E) Phase contrast images of wild-type mammary organoid preparations untreated or treated with HGF or HGF and TGF-ß1 shows TGF-ß-mediated inhibition of HGF-induced branching. Three representative images are shown for each condition. (F) Wnt5a^-/- organoids were untreated or treated with HGF or TGF-ß and HGF. Untreated Wnt5a^-/- cultures demonstrated increased branching relative to wild-type cultures. TGF-ß failed to inhibit branching in the absence of Wnt5a. Three representative images are shown for each condition. (G) Primary branching was quantified in wild-type and Wnt5a^-/- cultures untreated or treated with HGF or TGF-ß and HGF. In wild-type cultures treatment with TGF-ß resulted in a statistically significant decrease in branching. TGF-ß did not inhibit branching in Wnt5a^-/- cultures.