Mechanisms of cell death induced by RAD001 and vincristine alone and in combination. (A) Mice engrafted with ALL-1345 or ALL-1999 cells were treated with vehicle, vincristine, RAD001, or RAD001 plus vincristine for 24 hours and lysates prepared from recovered spleen cells. Lysates were subjected to Western blotting with anti-PARP, or anti–β-actin antibodies. (B) NALM6 cells were cultured with 2 μM RAD001, 1 nM vincristine, or the combination of both for 24 hours and cell lysates prepared. Lysates were subjected to Western blotting with anti-PARP, anticaspase 3, or anti-β-actin antibodies. (C) NALM6 cells were treated with the indicated doses of RAD001 for 24 hours, and cell lysates were analyzed for Beclin-1 and LC3 expression by Western blotting. β-actin is shown as a loading control for Beclin-1, and the ratio of LC3-II/LC3-I is indicated below the blots for LC-3. NALM6 or REH cells were treated with vehicle alone (Control), or 2 or 16 μM RAD001 for 24 hours. Cells were stained for acidic vacuoles using Lysosensor Blue. Representative fields of view are shown in panel D. Original magnification ×600. See “Immunofluorescence microscopy” for details. The proportion of cells containing acidic vacuoles (AV) (E) and the number of AV in each cell (F) are shown. Quantitation was obtained from the analysis of more than 120 cells for each condition in each of 2 separate experiments. The mean plus or minus SD of the independent experiments is shown for NALM6 cells.