Antimycin

Cytotoxicity assays were performed for metformin (Sigma-Aldrich), arsenic trioxide (Sigma-Aldrich), oligomycin (Sigma-Aldrich), antimycin (Sigma-Aldrich), and aTOS (Sigma-Aldrich) (Fig S3A). Experimental concentrations with low cytotoxicity were chosen and assayed for 24 h. All assays were performed in biological triplicates and analyzed for viability using flow cytometry, as described below.

β-Naphthoflavone (BNF), DMSO, catalase, ubiquinol, ADP, sodium succinate, NADH, cytochrome C, lauryl maltoside, oligomycin, 2,4-dinitrophenol (DNP), rotenone, antimycin, CH223191, proadifen, and resveratrol were obtained from Sigma Chemical Co. (St Louis, MO). ROS probes 2′,7′–dichlorofluorescin diacetate (DCFDA) and Amplex Red reagents were purchased from Abcam (Cambridge, MA) and Invitrogen, (Carlsbad, CA), respectively. Rat C6 glioma and COS cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and grown in DMEM/F12 or MDM2 media obtained from Invitrogen, (Carlsbad, CA). In all cases, cells were grown in culture medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at 5% CO₂, and 95% air (v/v), at 37°C in the incubator. In some cases, cells were also treated for 24–48 hrs with BNF dissolved in dimethylsulfoxide (DMSO; 25–50 μM) in the presence or absence of resveratrol (10 μM), Mito-CP (2 μM), AHR inhibitor CH223191 (25 μM), and CYP inhibitor proadifen (5 μM), whereas the control cells were treated with vehicle alone.

The metabolic profile was monitored in control U87MG cells or in cells subjected to AKAP1 silencing. Real-time measurements of OCR were made using an XF-96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Cells were plated in XF-96 plates (Seahorse Bioscience) at the concentration of 2 × 10⁴ cells/well and cultured for the last 12 h in DMEM, 10% FBS. OCR was measured in XF media (non-buffered DMEM medium, containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate), under basal conditions and in response to 5 μM oligoMycin, 1.5 μM of FCCP and 1 μM of AntiMycin and Rotenone (all from Sigma Aldrich). Indices of mitochondrial respiratory function were calculated from OCR profile: basal OCR (before addition of oligoMycin), ATP-linked OCR (calculated as the difference between basal OCR rate and oligoMycin-induced OCR rate) and maximal OCR (calculated as the difference of FCCP rate and AntiMycin+Rotenone rate).

Seahorse analysis was performed on an XFe96 Analyser from Agilent Technologies (Santa Clara, CA, USA). Cells were plated at a density of 2.0 × 10⁴ cells per well for each cell line 24 h prior to analysis. Cells were washed twice with unbuffered Seahorse medium (Agilent #102353-100 DMEM, Santa Clara, CA, USA, with or without 3 mM L-glutamine and with 10 mM glucose) prior to adding Seahorse medium containing drugs. Cells were treated with the drugs for 10 min prior to starting the assay, during which the drugs remained present. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured during subsequent injections of 2.5 µM oligomycin (ATP synthase inhibitor, Sigma #O4876, St. Louis, MO, USA), 50 µM 2,4-dinitrophenol (DNP; uncoupler, Sigma #D19850, St. Louis, MO, USA), 2 µM rotenone with 4 µM Antimycin A (complex I and III inhibitors, respectively, Sigma #R8875 and #A8674, St. Louis, MO, USA) and lastly, 100 mM 2-deoxyglucose (2-DG; glucose analog and hexokinase II inhibitor, Sigma #D8375, St. Louis, MO, USA); these are final concentrations in the wells. The OCR:ECAR ratio was calculated from basal OCR and ECAR corrected for background levels of OCR and ECAR after 2-DG injection. Spare capacity was calculated by subtracting the basal OCR from the maximal OCR.

DiBAC₄(3) was obtained from Thermo Fisher Scientific. Rotenone, antimycin, oligomycin B and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (all from Sigma-Aldrich, Dorset, UK) were dissolved in DMSO as 1, 1, 6 and 10 mM stock solutions, respectively. K⁺ channels inhibitors, glibenclamide (Sigma-Aldrich), penitrem A (Alomone labs, Jerusalem, Israel), tram34 (Sigma-Aldrich), apamin (Tocris Bioscience, Abingdon, UK) and XE991 (Tocris Bioscience) were also prepared in DMSO as 10, 1, 10, 1 and 10 mM stock solutions, respectively. Other chemicals were obtained from Sigma-Aldrich or VWR (Leicestershire, UK).

Cells were seeded and treated in the same manner for 45 min as for Annexin V/Hoechst cytometry assay. Prior trypsinization, cells were incubated with 20 nM Mitotracker Red (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial membrane potential (MMP) detection, 20 nM Mitotracker green (Thermo Fisher Scientific) for mitochondrial volume determination, 20 µM H2DCF-DA probe (Sigma Aldrich) for oxidative stress assessment, and 1 µM Fluo-4 AM (Thermo Fisher Scientific) for intracellular Ca²⁺ determination. As positive control, cells were stained with 100 μM TBHP, 10 μM antimycin and FCCP (Sigma Aldrich). After incubation, cells were trypsinized, centrifugated (3 min, 100× g, 4 °C) and transferred to a 96-well plate and directly analyzed by flow cytometry using the 488 ex 525/50 bp filter for detecting H2DCF-DA, Mitotracker green and Fluo-4 AM fluorescence and the 561 ex 586/15 bp filter for detecting Mitotracker Red fluorescence.