18 hours prior to the assay. Immediately following the addition of fresh medium, O2 consumption rate (OCR) and proton produc- tion, expressed as the extracellular acidification rate (ECAR), were quantified to obtain baseline levels of these processes. The next measurement was performed following the blockade of complex V by 1 mM oligomycin (injection A). This causes a build-up of protons across the inner mitochondrial matrix with subsequent loss of electron flow. The addition of FCCP (final concentration 300 nM; injection B) causes the protons on the outside of the inner membrane to be carried across to the mitochondrial matrix. The addition of 2-deoxyglucose (2-DG) (final concentration 100 mM; injection C) inhibits glucose uptake, glycolysis, and the generation of acetyl coenzyme A from pyruvate for utilization as an initial substrate for the TCA cycle. Finally, the injection of rotenone (final concentration 1 mM; injection D) inhibits complex I, leading to cessation of both electron flow and oxygen consumption. Experiments were performed by simultaneously measuring three to five replicates of each cell line. Relative effects were expressed as areas under the curve measurements that were generated by the manufacturer’s software. Measurement of ATP levels ATP levels were measured using the ATPliteTM Luminescence Assay System (Perkin Elmer, Waltham, MA) according to manufacturer’s instructions. 20,000–30,000 cells were grown overnight in 96-well plates and were exposed to 100 ml of medium with and without metabolic inhibitors at 37uC for the final 45 minutes prior to the assay. The concentrations of the inhibitors were the same as those used for the Seahorse assay described above. The half-life of ATP was determined by first growing the cells as described above. Warm medium containing both oligomycin and 2-DG was then added and the cells were incubated at room temperature. At various points thereafter, lysis solution was added to stop the reaction. Luminescence was measured as described above and the exponential line equation was used to calculate the half-life. Preparation of mitochondria from fibroblasts Approximately 107 freshly harvested fibroblasts were re- suspended in 0.5 ml of ice cold buffer 25 mM Tris-HCl, pH 7.5; 100 mM KCl; 0.4 M sucrose containing protease inhibitor cocktail (Sigma-Aldrich) and disrupted in a cell homogenizer (Isobiotec, Heidelberg, Germany) for 10 strokes on ice. The resultant homogenate was clarified by centrifugation at 5006 g for 10 min at 4uC, and the pellet was discarded. The mitochondria-rich supernatant was then centrifuged at 14,0006 g for 15 min. The pellet was washed twice with buffer A and re- suspended in same buffer at a final protein concentration of 5 mg/ ml. Samples were either analyzed immediately or stored at 280uC. Blue native gel electrophoresis (BNGE) Eight mg of digitonin (MP Biomedicals, Solon, OH) was dissolved in 200 ml of 30 mM HEPES buffer, pH 7.4, 150 mM potassium acetate and 10% glycerol, heated at 95uC, then cooled on ice. One mg of mitochondria, isolated as described above, was lysed by the addition of digitonin to a final ratio of 1:8 protein:digitonin. Following incubation on ice for 20 min, a Coomassie blue solution (5% Coomassie blue G250 in 750 mM 6-aminocaproic acid) was added (1/20 v/v), and the mixture was centrifuged at 14,0006g for 20 min at 4uC. The supernatant was then directly loaded onto a 3– 12% Native PAGE Novex Bis-Tris gel (Invitrogen), and subjected to electrophoresis at 80 V for 4 hours at 4uC in the buffer provided by the supplier. Following electrophoresis, gels were stained with Bio- Myc Influences Mitochondrial Dynamics Safe Coomassie G250 (Bio-Rad, Hercules, CA) for 30 min and exhaustively de-stained with water. Stained gels were scanned and the images analyzed for relative band density using AlphaEaseFC 2200 scanner and AlphaEaseFC software. For confirmation of native complex identities, individual bands were excised from the above gels, incubated in Laemmli SDS- sample buffer (Bio Rad) for 30 min at room temperature and electrophoresed on a Criterion pre-cast gel (Bio Rad) and electrophoresed at 80 V for 2 hours followed by silver staining (Sigma-Aldrich). Complex I and V gel in situ activity stain For Complex I activity measurements, the blue native gel was placed in 3–4 ml of 2 mM Tris-HCl, pH 7.4 buffer containing 2.5 mg/ml nitrotertrazolinum blue chloride and freshly added 0.1 mg/ml NADH. The gel was incubated at 37uC for 1–2 hours and then subjected to densitometric analysis. An average value from 3 gels was calculated. To quantify the ATPase activity of Complex V, gels were incubated in 3–4 ml of 34 mM Tris-glycine, pH 7.8, 14 mM MgSO4, 0.2% Pb(NO3)2 with freshly added 8 mM ATP for 3 hours at 37uC. Bands were quantified as for Complex I. Immunoblotting Cells were grown to approximately 90% confluency under standard conditions and then harvested by trypsinization. After washing twice in PBS, cell pellets were lysed in SDS-PAGE lysis buffer without b-mercaptoethanol, and protein concentrations were quantified using a BCA protein determination kit (Pierce, Rockford, IL). 5 mg of total protein lysate from each cell line was then resolved on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Inc., Bedford, NY) by electrophoretic transfer according to manufacturer’s instructions (Bio-Rad). Immunoblotting was performed as previously described [72]. The antibodies and the concentration at which they were used are listed in Table S1. The blots were developed using chemiluminesence (Thermo Fisher Scientific, Rockford, IL). Supporting Information Figure S1 Western analysis for Myc expression. 5 mg of whole cell lysates from myc+/+, myc2/2 and myc2/2wtMyc cells were used to perform Western analysis with the 9e10 anti-Myc antibody. b-actin is used as a loading control. (TIF) Figure S2 ECAR in rat fibroblasts. Extracellular acidifica- tion rates (ECARs) were calculated concurrently with OCR. ECAR is a surrogate measure of glycolysis and is expressed as a function of time. Inhibitors were injected at the times indicated by the arrows (injections: A-oligomycin, B-FCCP, C-2-DG, D- rotenone). A typical experiment, performed in triplicate wells is shown. The experiment was repeated on at least three occasions in replicates of four with similar results. (TIF) Figure S3 ATP half life. ATP levels were measured for the myc+/+ and myc2/2+wtMyc fibroblasts. The cells were incubated for the indicated times in the presence of 2-DG and oligomycin. A logarithmic curve was fit for each data set and the equation of the line was used to calculate the half life. Depicted is a representative experiment. (TIF) Antibodies used in this study. Table S1 (PDF) PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e37699