OSI-027 induces Puma- and Bim-dependent apoptosis. (A) Parental Jurkat cells and Jurkat variants lacking critical apoptotic pathway components (inset) were treated for 48 hours with OSI-027, stained with PI and analyzed as illustrated in Figure 5D. Error bars, mean ± SEM of 3 independent assays. (B,D) Twenty-four hours after transfection with control siRNA or siRNA targeting Bax or Bak (B) or Bim or Puma (D) along with plasmid encoding EGFP-histone H2B (to mark successfully transfected cells), Jurkat cells were harvested to assess target down-regulation or treated for 48 hours with the indicated concentration of OSI-027, stained with APC–annexin V, and analyzed by 2-color flow cytometry to assess annexin V binding in EGFP-histone H2B⁺ cells. Error bars, mean ± SEM of 3 independent assays. Insets, immunoblots (B) or qRT-PCR (D) demonstrating down-regulation of the targeted protein or message, respectively. (C,E) After Jurkat (C) and Jeko cells (E) were treated for 48 hours with diluent (lanes 1); OSI-127 at 1.25, 2.5, 5, 10 or 20μM (lanes 2-6); or 10nM rapamycin (lane 7) in the presence of the caspase inhibitor Q-VD-OPh (5 μM) to inhibit apoptosis,²²  whole cell lysates²⁸  were subjected to SDS-PAGE followed by immunoblotting with antibodies that recognize the indicated antigen. PARP1 served as a loading control. (F-G) After Jurkat cells were treated for 48 hours with diluent, OSI-027 at the indicated concentration or 10nM rapamycin in the presence of 5μM Q-VD-OPh, RNA was isolated and subjected to conventional RT-PCR or qRT-PCR. Noxa and GAPDH served as controls. Results in panel F, geometric mean of 3 independent experiments. (H) Beginning 24 hours after transfection with the indicated reporter construct, Jurkat cells were treated for 48 hours with diluent or OSI-027 at the indicated concentration in the presence of 5μM Q-VD-OPh and cell lysates were assayed for firefly luciferase. Error bars, mean ± SD of 4-6 independent experiments.