2 4 dinitrophenol

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 K_ATP channel currents and action potentials were recorded by whole-cell patch-clamp studies in hESC-VCMs at 37 °C using the EPC10 amplifier and Pulse/PulseFit software (HEKA, Germany) as previously described12 (link). Patch electrodes (3.0–5.0 MΩ) were filled with the internal pipette solution containing 120 mM potassium glutamate, 25 mMKCl, 1 mM ATP (magnesium salt), 10 mM EGTA, 0.5 mM MgCl₂ and 10 mM HEPES (pH 7.2). The external bath solution contained 140 mMNaCl, 5 mMKCl, 1 mM MgCl₂, 1 mM CaCl₂ and 10 mM HEPES (pH 7.4). Glibenclamide (Sigma-Aldrich), diazoxide (Sigma-Aldrich), 2, 4-dinitrophenol (Sigma-Aldrich) and P1075 (Santa Cruz Biotechnology) were dissolved in DMSO before they were added to experimental solutions. Sodium cyanide (Sigma-Aldrich), 5-hydroxydecanoic acid (Sigma-Aldrich) and HMR 1098 (Axon Medchem BV) were dissolved directly in external bath solution.