Effect of R5020 plus HGF on proliferation of epithelial cells. Murine mammary epithelial cells were suspended in collagen type I gels and cultured in (a) HGF alone (HGF, 50 ng/ml) or with HGF in combination with E2 (10 nM), R5020 (20 nM) or E2+R5020 (10 nM+20 nM) or (b) in FCM with or without R5020 or E2+R5020. 3H-thymidine incorporation into DNA was assayed after 3 days of culture. The data are expressed for suspensions in basal medium as 3H-thymidine incorporated per well and for HGF- and FCM-treated groups as fold increase over basal-medium control. *P = 0.05 that proliferation is greater in HGF+R5020 group than in HGF or HGF+E2. ** P = 0.01 that the fold increase in proliferation in suspensions in HGF+ E2+R5020 and FCM+ E2+R5020 is greater than in all other groups within the same experiment. (c) Phase-contrast photomicrographs of epithelial cell organoid morphology in collagen gel cell culture after 3 days in basal medium containing R5020, RU486, HGF, R5020+HGF, RU486+R5020, or RU486+R5020+HGF. ×100. Note appearance of lumens (L) and alveolar buds (AB) in R5020 and R5020+HGF-treated cultures, respectively, and long tubules (T) in HGF and RU486 +R5020 +HGF-treated cultures. No lumen or alveolar bud formation was observed in the presence of RU486. (d) Histological sections of three separate alveolar-like organoids obtained from cultures treated with HGF+R5020;-estradiol; FCM = fibroblast-conditioned medium; note presence of multiple lumens (L) within these structures. AB, alveolar bud; E2, 17β HGF = hepatocyte growth factor; T, tubule.