Autocrine epidermal growth factor receptor activation unnecessary for ERK1/2 stimulation of proliferation or apoptosis resistance. (a) Acini were grown for 10 days and then treated with diluent, with 100 nM 4-hydroxytamoxifen (4-HT) or with 100 nM 4-HT + 300 nM AG1478 (epidermal growth factor receptor (EGFR) inhibitor) and were immunostained with α-Ki-67 (green) and α-cleaved caspase 3 (red) antibodies and counterstained with Hoechst (blue). The acini shown are representative of 100 acini observed in three independent experiments. No more then 10 acini in the 4-HT-treated acini or 4-HT + AG1478-treated acini had greater then 10% of cells in the lumen stain positive for cleaved caspase 3 in any of the experiments. Bar = 75 μm. DMSO, dimethylsulfoxide. (b) Raf:ER-expressing and H2B:GFP-expressing MCF-10A cells were plated at a 1:1 ratio and grown in three-dimensional culture for 13 days with diluent, with 100 nM 4-HT in the absence of EGF, or with 100 nM 4-HT, no EGF and 300 nM AG1478 (EGFR inhibitor). The nuclei of Raf:ER acini are seen with Hoechst (blue) and the nuclei of the H2B:GFP-MCF-10A cells are visualized with green fluorescence protein (GFP) (green) overlaying Hoechst (blue). Bar = 50 μm.