Pharmacologic inhibition of PI3K signaling reduces Mcl-1 expression and sensitizes B-NHL cells to rituximab-induced apoptosis in vitro and rituximab treatment in vivo. (A) Immunoblot analyses of rituximab-resistant B-NHL cells treated with the PI3K inhibitor LY294,002 (10 μM) or vehicle (−) using the indicated primary antibodies. Note the down-regulation of endogenous Mcl-1 expression in PTEN-negative Jeko-1 and HT B-NHL cells by the PI3K inhibitor. (B) Rituximab-resistant B-NHL cells were incubated for 48 hours with cross-linked rituximab (Rc), the PI3K inhibitor LY294,002 (LY, 10 μM), or both. The fraction of cells with apoptotic DNA fragmentation was quantified flow cytometrically; mean values plus SD of 3 independent experiments are shown. (C) Kaplan-Meier plots of symptom-free survival of NOD/SCID mice after intravenous inoculation of 10⁷ HT cells. Starting on day 5, 2 groups of mice received intraperitoneal injections of rituximab (HT + R, gray dots, solid line) or vehicle (HT, gray boxes, dashed line). A third group received intraperitoneal injections of the PI3K inhibitor LY294,002 (HT + LY, black triangles, dashed line), whereas the fourth group was treated with LY294,002 in combination with rituximab (HT + R + LY, black diamonds, solid line); 8 mice were treated in each group. Note that LY294,002 successfully sensitized the mice toward rituximab treatment.