The mTOR pathway is activated in all mouse T-ALL cell lines examined. (A) Primary mouse T-ALL tumors exhibit activation of Akt and mTOR pathways. Thymomas from vehicle-treated mice were harvested at killing and tumor sections were fixed in 10% buffered formalin. Sections were then stained with antibodies to phospho-S6 ribosomal protein (p-S6RP) or phospho-Akt (Ser473). Scale bars represent 50 μM. (B) Reduced levels of mTOR substrates are observed when mouse T-ALL lines are treated with GSI. Primary murine leukemic lines were treated with 1 μM MRK-003 or DMSO for 48 hours. MTOR activity was assayed by immunoblotting cell lysates with anti–phospho-p70 S6 kinase or phospho-S6 ribosomal antibodies (no. 9205, no. 2215; Cell Signaling Technology). p70 S6 kinase and S6 ribosomal were used as loading controls (no. 9202, no. 2217; Cell Signaling Technology). (C) Notch1 regulates Akt activity in some mouse T-ALL lines. Primary murine leukemic lines were treated with 1 μM MRK-003 or DMSO for 48 hours. Akt activity was assayed by immunoblotting cell lysates with anti–phospho-Akt Ser 473 antibody (no. 9271; Cell Signaling Technology). Akt was used as a loading control (no. 9272; Cell Signaling Technology). Fold reductions in kinase activity were determined by densitometry and represent ratios (phospho/total) normalized to DMSO-treated samples.