Implication of PI3K/AKT pathway in mTOR activity. (A) Phosphorylation of AKT was evaluated in FL cell lines and compared to B cells isolated from healthy donors (BL) and from B-CLL patients (B-CLL) by Western blotting using anti-phospho-AKT (Thr308) antibody. AKT was used as control of protein expression. Results are representative of 3 independent experiments. (B) PI3K-dependent activation of mTOR was evaluated in Karpas-422 and RL cells treated or not with wortmannin (WT) or LY294002 for 30 minutes using antiphospho-p70S6K (Thr389) antibody. p70S6K was used as control of protein expression. CT corresponds to untreated FL cells and results are representative of 3 independent experiments. (C) The effect of Syk inhibition on PI3K activity was evaluated on RL cells. PI3K activity assay was performed as indicated in “Materials and methods” with cells treated 2 hours with 50 μM piceatannol. The PIP₃ products of PI3K activation are indicated. Negative control corresponds to cell lysate incubated without p85 antibody. Results are representative of 3 independent experiments. (D) The effect of Syk inhibition on AKT phosphorylation was evaluated on Karpas-422 and RL cell lines. Cells were treated with piceatannol at various doses for 2 hours and AKT phosphorylation (Thr308) was evaluated by Western blotting. Results are representative of 3 independent experiments.