Bongkrekic acid

GSK1059615 was purchased from Adooq Bioscience (Wuxi, China). Gemcitabine and mPTP blockers sanglifehrin A, cyclosporin A and bongkrekic acid as well as wortmannin, LY294002 and perifosine, were provided by Sigma (Shanghai, China). MK-2206, rapamycin and AZD-2014 were purchased from Selleck (Shanghai, China). Antibodies for phospho-PI3K p85 (Tyr458) (#4228), PI3K p85 (#4257), p-AKT (Ser 473, #9271), AKT1 (9272), p-p44/42 MAPK (p-ERK1/2, #9101), ERK1/2 (#9102), p-S6K1 (Thr-389, #9205), mTOR (#2983) and p-mTOR (Ser-2448, #2971) were obtained from Cell Signaling Tech (Shanghai, China). Antibodies for tubulin, adenine nucleotide translocator-1 and cyclophilin-D were obtained from Santa Cruz Biotechnology (Shanghai, China).

Sodium sulfate (Na₂SO₄), 2-(N-morpholino)ethanesulfonic acid (MES), tris(hydroxymethyl)-aminomethane (Tris), sodium dodecyl sulfate (SDS) ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) were purchased from Carl Roth GmbH & Co. K.G. (Karlsruhe, Germany). Hexane, hexadecane, dimethyl sulfoxide (DMSO), purine nucleotides adenosine tri-, di- and guanosine triphosphate (ATP, ADP, GTP), agarose, carboxyatractyloside (CATR) and bongkrekic acid (BKA) were purchased from Sigma-Aldrich Co. (Vienna, Austria). 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cardiolipin (CL) came from Avanti Polar Lipids Inc (Alabaster AL, USA) and chloroform from Sanova Pharma GesmbH (Austria). Arachidonic acid (AA) was purchased from Larodan (Biozol, Eching, Germany).

Agarose (#3810), KCl (#6781), Na₂SO₄ (#8560), 2-(N-morpholino)ethanesulfonic acid (MES, #4256), sodium dodecyl sulfate (SDS, #0183), tris(hydroxymethyl)-aminomethane (Tris, #AE15), chloroform (#AE54) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, #3054) were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Hexane (#296090), hexadecane (#296317), palmitic acid (#P0500), stearic acid (#S4751), arachidic acid (#A3631), linoleic acid (#L1376) and arachidonic acid (#A3611), dimethyl sulfoxide (DMSO, #472301), the purine nucleotides adenine and guanine tri-, di-, and mono-phosphate (ATP, #A2383; ADP, #A2754; AMP, #01930; GTP #G8877; GDP, #G7127; and GMP, #G8377), carboxyatractyloside (CATR, #C4992) and bongkrekic acid (BA, #B6179) were purchased from Sigma-Aldrich (Vienna, Austria). 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, #850375P), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, #850725P) and cardiolipin (CL, #710335P) came from Avanti Polar Lipids Inc. (Alabaster, AL, USA).

MAPK Family Antibody Sampler Kit (#9926) and Phospho-MAPK Family Antibody Sampler Kit (#9910) were purchased from Cell Signaling Technology (Beverly, MA). Anti-CEP-1 (cC-18) (sc-135460) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Paraquat (PQ), Cyclosporin A (CsA), Bongkrekic acid (BA) and 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) were from Sigma. N-acety-L-cysteine (NAC) was purchased from TCI (Shanghai, China). Total Antioxidant Capacity Assay Kit with the ABTS Method (S0119), Fluo-3 AM (S1056), Hoechst 33342 (C1022), Mitochondrial Membrane Potential Assay Kit with JC-1 (C2006), Enhanced BCA Protein Assay Kit (P0010S) and Superoxide dismutase (SOD, S0088) were obtained from Beyotime (Shanghai, China). Acridine orange (AO) was from Dingguo Changsheng Biotechnology (Beijing, China). H₂DCF-DA (D399), MitoSox (M36008), Mitotracker red (M7512), ATP determination kit (A22066) and SYTO 12 (S7574) were purchased from Molecular Probes (Eugene, Oregon, USA). PVDF membranes and ECL plus detection kit were obtained from Millipore (Bedford, MA, USA).

The imaging procedure followed a previously described method [18 (link)]. Cells were seeded on poly-d-lysine-coated glass coverslips and imaging was performed in Hank’s balanced salt solution (HBSS, Gibco, New York, NY, USA) at room temperature. To track changes in mitochondrial membrane potential, the fluorescent probe tetramethylrhodamine, methyl ester (TMRM, Invitrogen, Waltham, MA, USA) was used. Cells were incubated with 20 nM TMRM for 15 min in the dark at room temperature. Recordings were performed in the presence of 20 nM TMRM.
To induce mitochondrial Ca²⁺ overload and subsequent mPTP, ferutinin at a concentration of 20 μM was used. RI images (holographic reconstructions) and TMRM signals were acquired every 15 s using a 3D Cell Explorer-fluo (Nanolive, Tolochenaz, Switzerland) equipped with a 60× objective. At the end of each experiment, 10 μM of the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, Sigma Aldrich, St. Louis, MO, USA) was added to induce complete depolarization of mitochondria, allowing for the determination of the minimal TMRM signal for subsequent normalization and analysis.
Cyclosporin A (CSA, Sigma Aldrich, St. Louis, MO, USA) and bongkrekic acid (BA, Sigma Aldrich, St. Louis, MO, USA), inhibitors of mPTP and ANT, respectively, were used to explore the regulation of the mitochondrial permeability transition.

C2C12 myoblasts obtained from the Cell Resource Center (Peking Union Medical College) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, GE) and 100 μg/mL penicillin/streptomycin antibiotics in an incubator with 5% CO₂ at 37 °C. Recombinant mouse full-length APN was synthesized by Gencreate (Wuhan, China) and dissolved in phosphate-buffered saline (PBS) at a concentration of 500 μg/mL. Hydrogen peroxide (H₂O₂) was purchased from Solarbio (Beijing, China). Bongkrekic acid (BA) was purchased from Sigma. Chloroquine diphosphate (CQ) was purchased from InvivoGen.