(A–D) Tumor sections from Akt-dependent melanomas expressing a control shGFP (A) or shNotch1 (B) were stained with anti Ki67, and were counterstained with DAPI, to label proliferating cells. (C and D) Adjacent sections stained with anti–cyclin D1 antibody. (E) Quantification of proliferating cells in A and B, shown as percent positive cells in 5 microscopic fields from 3 different tumor sections per group. (F) Melanocytes expressing either shGFP or shNotch1 were grown in culture and counted every 3–4 d. (G) Western blot analysis for Notch1-N^IC, β-catenin, and cyclin D1 of cells transfected with shGFP or shNotch1. β-Actin was used as loading control. (H–K) Tumor sections from cells expressing shGFP (H) or shNotch1 (I) were stained for Notch1-N^IC. (J and K) Adjacent sections were stained with anti–cleaved caspase-3. (L) Quantification of cell death under stringent hypoxia (0.5% O₂), measured as positivity to annexin V/propidium iodide. (M) Western blot analysis for Notch1-N^IC and cleaved caspase-3 (c-casp3) on cells grown in normoxia or stringent hypoxia for 48 h. Glut-1 was included as an indicator of the hypoxia treatment. β-Actin was used as loading control. Data in E, F, and L are mean ± SD. *P < 0.05 versus respective shGFP control, Student’s t test. Scale bars: 25 μm.