Proliferation and mTOR pathway activation are partially resistant to PI3K inhibition in r1ΔT/r2n T cells. (A) Purified T cells were treated for 15 minutes with the indicated inhibitors and then stimulated for 48 hours with platebound anti-CD3 alone or anti-CD3 plus CD28. Proliferation was measured by cell-cycle analysis, with the percentage of cells in S and G2 phases combined in the analysis. The mean ± SD of 4 experiments is shown. Inhibitor concentrations were 50 nM wortmannin; 10 μM LY294002, 424 (AS252424), and 850 (AS604850) (GE Healthcare Life Sciences, Piscataway, NJ); and 10 ng/mL rapamycin. (B) Immunoblot analysis for phosphorylation of S6 in purified T cells after 18 hours with indicated stimulation conditions. “Normalized units” refers to the relative band intensities of phospho-S6 compared with β-actin, calculated using ImageQuant. To compensate for its short half-life, wortmannin was added every 6 to 12 hours in both the proliferation and phospho-S6 experiments.
