Treatment of cancer cells with 2DG and ABT-263/737 We tested several conditions such as the length of pre- incubation and the concentration of deoxyglucose in the media (Fig. S2 and data not shown), and developed the following protocol. To cells cultured in appropriate medium, add 2DG at a molar ratio 0.5:1.0 2DG to glucose, and incubate the cells for three hours before ABT addition. In DMEM that contains 2 g/L of glucose, we typically used 10 mM 2DG, while in high glucose medium, we used 20 mM 2DG. We regularly used 1 mM ABT- 263/737 for the 2DG-ABT combination for cancer cells and 3 mM for MEFs. For a single-agent application, we used 10 mM ABT for cancer cells and 30 mM ABT for MEFs. Western blotting and immunoprecipitation We usually run either 12.5% or 4–20% gradient gels for Western Blots, except for detecting loss of long isoforms for Opa1 (8% SDS-PAGE gels), and separating Bak protein from IgG Light Chain (15% SDS-PAGE gels). The following antibodies were used for Western Blots: anti-Opa1 mouse mAb from Transduction Lab (Cat. 612606), anti-Cyt C mouse mAb from BD Bioscience (Cat 556433), anti-Bid mouse mAb from Santa Cruz (5C9) 0.1 mg/ml, anti-Bid Rabbit Polyclonal Ab from Thermo Scientific (Cat PA1-84831), anti-Bak NT Rabbit polyclonal Ab from Millipore, anti-Bax and anti-Mcl-1 Rabbit Polyclonal Abs were previously described [Krajewski, et al 1994 Am. S. Pathol 145– 515; Krajewski S et al 1995 Cancer Res 55: 4471], anti-Grp78 Rabbit Polyclonal Ab from ABR (Cat. PA1-014), anti-PKM2 antibody gifted from the Lewis Cantley laboratory. To immuno- precipitate of Bak and Mcl-1 proteins from PPC-1 cells, we used mitochondria-enriched fraction [20], re-suspended in buffer containing 1% Triton X-100 as described [20]. To immunopre- cipitate Bak and Mcl-1 proteins, antibodies against them (anti-Bak NT Rabbit polyclonal Ab from Millipore, and Anti-Mcl1 Rabbit Polyclonal Ab from Krajewski) were conjugated to Protein G- Sepharose, mixed with lysates, and incubated overnight on a rotating platform. To separate Bak from IgG Light Chain, we run immunoprecipitates in 15% SDS-PAGE. For detection of Mcl-1, we run precipitates in 12.5% SDS-PAGE. Immunofluorescence microscopy Cells were treated as described [31]. Briefly, cells were fixed in 3.7% formaldehyde for 20 min, washed in PBS, and permeabi- lized with 0.5% Triton X-100. Nonspecific binding was blocked by incubating cells in 0.2% gelatin/PBS for 30 min. Cytochrome c staining was performed using a primary antibody against cytochrome c (clone 6H2.B4, Cat 556432, BD Science) and a fluorescent secondary antibody conjugate, goat anti-mouse IgG, AlexaFluor 568 (Molecular Probes). Images were captured by a MRC 1024 SP BioRad Laser Point Scanning Confocal Micro- scope. For Bax translocation studies, we used anti-Bax Rabbit Polyclonal Ab [Krajewski, et al 1994 Am. S. Pathol 145–515; Krajewski S et al 1995 Cancer Res 55: 4471]. Electron microscopy Cells were prepared and the electron microscopy was per- formed following the procedure described in ‘‘Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells’’, [Ju WK, Kim KY, Lindsey JD, Angert M, Patel A, Scott RT, Liu Q, Crowston JG, Ellisman MH, Perkins GA, Weinreb RN. Mol Vis. 2009;15:120-34.] Bid siRNA From Qiagen Cat. No SI02661911 (59-AGACAAUGUUAAA- CUUAUATT-39 & 59-UAUAAGUUUAACAUUGUCUTT-39) 2DG-ABT Combination Therapy HeLa and PPC-1 cells were transfected with siRNA above and using Lipofectamine 2000 (invitrogen) using the manufacture’s protocol. We used mock transfection for controls. For both cells, transfections were done twice, 24 hours apart. Cells were used a day later for western blots and apoptosis assays. Apoptosis assay RS(4;11) cells were stained with Annexin V-FITC (Invitrogen PHN1008) and analyzed by FACSort (BD). HeLa cells were stained with Annexin V-APC (Invitrogen A35110) and analyzed by BDFACSCanto according to the manufacture’s instruction. Yoav Alterman and Shinichi Kitada at Sanford-Burnham Medical Research Institute performed FACS analysis. ATP measurement ATPlite solution (10 mL for 200 mL cell culture) was added, and a luminomitor (Monolight 3096; BD Biosciences) was used to measure luminescence. Caspase assay Cells grown to 80% confluency in 6 well dishes (diameter 34.9 mm) were washed in PBS and then incubated in appropriate media (e.g., DMEM with 12 mM glucose for HeLa cells). 5– 10 mM 2DG was added to the indicated samples, and 3 hours later, 1 mM ABT-737 was added to the indicated samples. 3 hours later, cells were (1) washed once in PBS, (2) incubated in 0.05% trypsin, 0.2 g/L EDTA in Hank’s Balanced Salt, (3) diluted in 106 volume DMEM containing 10% serum and trypsin inhibitor, (4) cells were span-down by centrifugation), (5) washed once with PBS, (6) pellets were re-suspended in 100 mL lysis buffer [20 mM Hepes, pH 7.4, 0.5% Triton X-100, 0.5 mM EDTA, and 150 mM NaCl] , (7) incubated on ice for 10 minutes, and then centrifuged at 10,000 g to remove the insoluble fraction. 10 mL of supernatant was diluted in 100 mL buffer [50 mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 0.1% CHAPS, 10 mmol/L DTT, 1 mmol/L EDTA, and 10% glycerol], containing 100 mmol/L Ac-DEVD-AFC and caspase activities were measured by a spectrofluorimeter at 37uC taking measurement for 20 min. Increases in relative fluorescence units [RFU] per minute were determined from the graph. Cell fractionation Cell were first washed in PBS twice, then re-suspended in buffer containing 10 mM Hepes (pH 7.4) 10 mM KCl, 1 mM EDTA, 1 mM EGTA and 250 mM trehalose, at one million cells per 0.5 ml. They were incubated on ice for 30 min. Cells were dounce or needle homogenized until a small sample of cells showed roughly 40% trypan blue positive. Cells were centrifuged at 10,000 g for 5 minutes for three times, each time collecting the supernatant. The supernatant was almost exclusively cytosolic fractions. Statistical analysis Statistical analysis was performed at Graphpad, http://www. graphpad.com/quickcalcs/ttest1.cfm?Format=C. Mouse xenograft Mice were maintained under specific pathogen-free conditions in accordance with guidelines by the Association for Assessment and Accreditation of Laboratory Animal Care International. CCHMC is credited through the Animal Welfare Assurance (#A3108-01). All animal experiments were carried out in strict accordance with animal protocol #8C03021 that was approved PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24102