Raf:ER activates AKT in organotypic culture. Raf:ER three-dimensional cultures were grown for 10 days, treated with diluent or 100 nM 4-hydroxytamoxifen (4-HT) and grown for an additional 48 hours. Acini were immunostained with both α-phospho-AKTS473 (red, top panels) and α-phospho-ERK1/2 (green, bottom panels) and counterstained with Hoechst (blue). The same acini are shown in the upper and lower panels of each treatment condition to demonstrate the relationship between ERK1/2 activation and AKT phosphorylation. There is some nonspecific staining of the Matrigel surrounding acini in the phospho-ERK1/2 images. Bars = 150 μm.