Antimycin

Hela cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (Sigma-Aldrich). Cells were incubated in a humidified atmosphere at 5% CO₂ and 37 °C. HeLa cells were transfected using Fugene (Promega) or PEI MAX 40000 (24765, PolySciences, Inc., Warrington) according to the manufacturer’s recommendations. HeLa cells were treated with 0 to 50 µM carbonyl cyanide m-chlorophenyl hydrazine (CCCP – Sigma-Aldrich), 10 µM oligomycin (Enzo Lifesciences), 1 µM antimycin (Enzo Lifesciences), 100 nM bafilomycin (Sigma-Aldrich) or 1 mM 1,2-dimethyl-3-hydroxy-4(1 H)-pyridon (deferiprone - DFP for the indicated time (Sigma-Aldrich).

C2C12 cell mitochondrial respiration, basal respiratory capacity, coupling efficiency, proton leakage, and spare respiratory capacity were profiled by an XF24 metabolic flux analyzer (Seahorse Bioscience, Billerica, MA, USA). Furthermore, the oxygen consumption rate (OCR) was assessed in C2C12 cells that had been induced to undergo differentiation while being continuously treated with the ATP synthase inhibitor oligomycin (Wako Pure Chemicals), the uncoupling agent carbonyl cyanide p-(trifluoromethoxyl)-phenyl-hydrozone (FCCP; StressMarq Biosciences Inc., Victoria, BC, Canada), and a mitochondrial complex inhibitor rotenone (Sigma-Aldrich)/antimycin (Enzo Life Sciences, Plymouth Meeting, PA, USA). Fatty acid β-oxidation was evaluated by measuring the OCR when differentiated C2C12 cells were treated with bovine serum albumin–conjugated palmitate after 1 h of equilibration in Krebs-Henseleit buffer containing 0.5 mM carnitine (Tokyo Chemical Industry, Tokyo, Japan) and 2.5 mM glucose.