Activation of Raf:ER induces pre-invasive epithelial growth in cultured acini. (a) Raf:ER-MCF-10A cells plated for three-dimensional culture were grown for 10 days. At day 10, diluent or 100 nM 4-hydroxytamoxifen (4-HT) was added and cultures were grown for an additional 5 days. Differential interference contrast images are shown. Bars = 150 μM. (b) The lysates of day 10 acini treated for 48 hours with diluent or 100 nM 4-HT were immunoblotted with α-phospho-ERK1/2 (top) and α-ERK1/2 antibodies. (c) Raf:ER-MCF-10A cells were grown as described in (a). Confocal cross-sections of acini immunostained with α-Ki-67 (green) and α-cleaved caspase 3 (red) and counterstained with Hoechst (blue) are shown. Bars = 50 μm. (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.