To confirm that the increase in sensitivity was not restricted to established cell lines, primary cultures were established from cells collected from four patients diagnosed with brain tumor (histologically confirmed glioblastoma). protein concentrations in the supernatants were determined. Equal amounts of protein extracts were incubated overnight with primary antibody. Afterward, Dynabeads Protein G (Invitrogen) were added for 2 hours. Supernatant (nonimmunoprecipitated fraction) was recovered by magnetic separation, and G-protein beads (immunoprecipitated fraction) were washed with ice-cold CHAPS lysis buffer. The beads were boiled in SDS sample buffer. The presence of immunocomplexes was determined by Western blot analysis. Bax/Bak Conformational Change. To analyze conformational changes of Bax and Bak, cells were lysed in CHAPS lysis buffer (1% CHAPS, Sulbutiamine 10 mM HEPES, 150 mM NaCl, and protease inhibitors) and immunoprecipitated in lysis buffer by using 500 for 10 minutes. After centrifugation, the pellet was washed with isotonic buffer and further extracted with ice-cold detergent (1% CHAPS) in isotonic buffer containing protease Sulbutiamine inhibitors for 60 minutes at 4C to release membrane- and organelle-bound proteins, including mitochondrial cytochrome 0.05. Results Antiproliferative Activity of BKM120 in a Panel of Glioma Cell Lines. In the present study, to investigate the growth inhibitory effect of BKM120, we cultured Sulbutiamine glioma cells with various genotypic features (see 0.005, compared with vehicle-treated cells. (B) LN18 and LNZ308 cells were treated with BKM120 for the indicated concentrations/duration. Cell extracts were subjected to Western blot analysis with indicated antibodies. Total AKT served as loading control. (C) Cells were seeded at 60% confluence, allowed to attach overnight, and treated with the indicated concentrations of BKM120 for 24 hours. Cell cycle analysis using propidium iodide staining was performed as described under 0.005, compared with vehicle-treated cells. NVP-BKM120 Promotes ABT-737CInduced Toxicity in a Caspase-Dependent Manner. In our recent studies, we have demonstrated that ABT-737 induces minimal growth inhibition in glioma cell lines; however, simultaneous treatment with the proteasomal inhibitor bortezomib (Premkumar et al., 2012) or survivin inhibitor YM-155 (Jane et al., 2013) enhanced ABT-737Cinduced cytotoxicity in a synergistic manner. Because inhibition of apoptosis by Akt has been characterized in many cancer cell systems, including glioma, and Akt levels affect ABT-737 sensitivity (Premkumar et al., 2012), we questioned whether ABT-737 may be best used in combination with BKM120. First, to quantify the effects of the inhibitor combinations on apoptosis, LN18, LNZ308, LN229, T98G, and U87 cells treated with compounds for 24 hours were stained with annexin V and PI, and analyzed by flow cytometry. Three experiments were performed in duplicate with similar results. A representative bar graph is documented in Fig. 2A. Single-agent ABT-737 or BKM120 resulted in only modest annexin V/PI staining. On the other hand, cotreatment with ABT-737 and BKM120 enhanced annexin V/PI sensitivity. In accordance with the annexin V/PI analysis, the combination of ABT-737 and BKM120 strongly induced activated caspase-8, -7, -3, and PARP in LN18 (PTEN wild type) and LNZ308 (PTEN deleted) cell lines. The combination of BKM120 and ABT-737 strongly induced caspase-8 processing with 18-kDa cleavage product and caspase-3 with 19-, 17-, and 12-kDa cleavage products. Although dose-dependent cleavage of PARP is seen to a limited extent with BKM120 alone, there is substantially more dramatic cleavage (89-kDa fragment) of PARP with the combination of BKM120 and ABT-737 (Fig. 2B; Supplemental Fig. 2). Similar results were obtained for T98G, U87, and LN229 cell lines (data not shown). In addition to viability, colony-forming ability was confirmed by clonogenic growth assay in four different glioma cell lines. Neither ABT-737 nor CDK4 BKM120 alone resulted in a significant reduction of viable cells; however, cotreatment of ABT-737 + BKM120 significantly inhibited colony-forming ability (Fig. 2C). Sulbutiamine Next, to further examine the role of the caspase signaling pathway, cells were treated with 0.001, compared with BKM120 or ABT-737 as a single agent versus combination of ABT-737 plus BKM120. (B) LN18 and LNZ308 cells were treated with ABT-737 (2.0 (Supplemental Fig. 2). (C) Human glioma cells were exposed to the indicated concentrations of BKM120 with or without ABT-737 for 24 hours. On the following day, the media were changed, complete media were added, and cells were grown for an additional 14 days in Sulbutiamine the absence of inhibitors. Control cells received equivalent concentrations of vehicle (DMSO). Colonies were fixed and stained as described under (Supplemental Fig. 3). Bar chart data represent mean S.D. of three independent experiments carried out in triplicate. ** 0.005; ns, not significant. Sensitization of Primary Cultures of Cells Derived from Patients with Glioma. To confirm that the increase in sensitivity was not restricted to established cell lines, primary cultures were established from cells.