Activation of other PI3 kinase–dependent signaling proteins. (A) Cells from patient collections were thawed, washed, and either immediately lysed in 1% Triton X-100 lysis buffer (lanes 1, 7, 13) or incubated in cytokine-free, serum-free medium for 4-16 hours in the absence of added compounds (lanes 2, 8, 14), in 0.1% DMSO (lanes 3 and 9), 0.1% ethanol (lanes 5 and 11), 25 μM LY294002 in DMSO (lanes 4, 10, 15), or 10 nM RAD001 in ethanol (lanes 6, 12, 16). After incubation, cells were lysed and 100 μg protein per lane analyzed using SDS-PAGE and Western blotting. Protein phosphorylation and expression were examined using indicated antibodies. In top panel, upper band is the p85 isoform of S6 kinase, which is constitutively expressed and phosphorylated in all samples. This band acts as a loading control. The blot was cut horizontally and the lower portion probed for phospho–4EBP-1 (lower panel). The upper panel was stripped and reprobed for phospho-Akt (middle panel). F indicates fresh; G, growing; D, DMSO 0.1%; L, LY294002 25 μM; E, ethanol 0.1%; and R, RAD001 10 nM. (B) Leukemic samples were thawed and incubated in serum-free medium with cytokines for 48 hours in varying concentrations of RAD001 as shown. Relative cell survival was measured using an XTT assay. ▪ indicates patient no. 111; ○, patient no. 88. (C) Cell line U937 cells were incubated in the presence of 10 nM RAD001 alone (▵),Ara-C alone (□), or Ara-C with 10 nM RAD001 (•). Relative cell survival was measured after 48 hours using an XTT assay. Error bars indicate SDs from 3 experiments.