Cell cycle progression in Raf:ER-stimulated acini requires phosphoinositide-3 kinase activity. (a) (Top panels) Acini were immunostained with α-cyclin B1 (green) or α-p27 (red) and counterstained with Hoechst (blue). (Bottom panels) Acini were immunostained with α-phospho-AKTS473 (green) or α-p27 (red) and counterstained with Hoechst (blue). (b) Acini were grown for 10 days and then treated with diluent, with 100 nM 4-hydroxytamoxifen (4-HT) or with 100 nM 4-HT and inhibitor. U0126 (10 μM), LY294002 (50 μM) and AG1478 (300 nM) were used. Fresh media with, diluent, with 4-HT or with 4-HT and inhibitor was added after 24 hours of primary treatment, and acini were cultured for another 24 hours (48 hours total treatment time). (Upper panels) α-c-Fos (red) and α-phospho-AKTS473 (green). (Lower panels) α-p27 (red) and α-cyclin B (green). All samples were counterstained with Hoechst (blue). Bar = 30 μm. DMSO, dimethylsulfoxide. (c) The number of acini containing at least two phospho-AKTS473-positive cells was scored. Data are the mean ± standard error of the mean of 100 acini scored in three independent experiments. (d) The percentage of acini containing three or more Ki-67 cells was quantified. Data are the mean ± standard error of the mean of 100 acini counted in three independent experiments.