Phosphoinositide-3 kinase activity is not necessary for reduced cell–cell adhesion or MLC2 (Ser-19) phosphorylation. (a) Raf:ER acini were treated with diluent, with 100 nM 4-hydroxytamoxifen (4-HT) or with 100 nM 4-HT and inhibitor as indicated after at least 10 days of growth in organotypic culture. Acini were immunostained with either anti-E-cadherin (red, top panels) or anti-phospho-MLC2 (Ser-19) (red, bottom panels) antibodies and counterstained with Hoecsht (blue, nuclei). Results are representative of at least three independent experiments. Bar = 30 μm. DMSO, dimethylsulfoxide. (b) Green fluorescence protein (GFP)–Raf:ER acini were cultured as described in (a). Acini were immunostained with anti-phospho-MLC2 (Ser-19) antibody (red, top panels) and counterstained with Hoecsht (blue). The expression of GFP-Raf:ER (green) is shown in the bottom panels. Bar = 50 μm.