RAD001 arrests ALL cells in G₀/G₁ phase of the cell cycle. Mice were treated with vehicle, RAD001, vincristine, or RAD001 plus vincristine for 1 or 7 days. (A) ALL cells isolated from spleens were labeled with 7-amino-actinomycin D and analyzed by flow cytometry. Cell-cycle profiles of ALL-1345 are shown as representative examples. (B) The percentage of cells in each stage of the cell cycle after 1 day of treatment as indicated is shown. The mean plus or minus SD of the percentage of cells in each cell phase from replicate mice is shown (n ≥ 4). (C) Hematoxylin and eosin–stained sections from femurs of mice engrafted with ALL-0398 are shown with examples of mitotic () and apoptotic () figures. Original magnification ×200 (details in “Histology”). Higher-power examples of mitotic and apoptotic figures produced by a further 20× digital enlargement are shown below. Larger images available in Figure S3. (D) Effect of RAD001 and/or vincristine treatment on the phosphorylation status of Rb and the levels of cdks 4 and 6 after 1 day of treatment of NOD/SCID mice engrafted with ALL-1345 or ALL-1999. The numbers below the plots indicate the ratio of cdk4 and 6 to β-actin.