MEFs were generated by harvesting Ant1−/− Ant2fl/fl Ant4−/− embryonic day 10.5 embryos. After the removal of the internal organs and the head, the remainder of the bodies were passed through a 25-gauge needle and plated on 10-cm2 tissue culture dishes and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) containing 10% bovine growth serum (BGS, Thermo Fisher Scientific), penicillin-streptomycin (Thermo Fisher Scientific), and nonessential amino acids (Invitrogen). After two passages, the MEFs were subjected to simian virus 40 (SV40) large T antigen immortalization by infecting with an SV40 T antigen–expressing lentivirus (Addgene, plasmid 22298, pLenti CMV/TO SV40 small + large T). Once immortalization was achieved, cells were treated with an adenovirus expressing both green fluorescent protein (GFP) and Cre recombinase (AdCre) for Ant2 gene deletion. Before infection, these MEFs were switched to “Rho 0” medium, which was the standard DMEM described above supplemented with uridine (50 μg/ml; Sigma-Aldrich) and 1 mM sodium pyruvate (Thermo Fisher Scientific). After infection, MEFs were subjected to fluorescent-activated cell sorting to isolate individual GFP-positive cells that had AdCre virus infection, which established clonal Ant-triple-null MEFs. Validation of the loss of ANT2 protein was determined by Western blot analysis. After cell selection, we were able to culture Ant-deleted MEF lines in high-glucose DMEM (4.5 g/liter glucose; Thermo Fisher Scientific) supplemented with 10% BGS (Thermo Fisher Scientific) and nonessential amino acids (Invitrogen). To determine if the Ant-triple-null MEFs relied on mitochondria-produced ATP for survival, we cultured the MEFs in glucose-free media (Thermo Fisher Scientific) with the addition of galactose (4500 mg/ml; Sigma-Aldrich), which caused their death within 24 hours, as measured with the Muse Cell Analyzer (MilliporeSigma) and the Muse Count & Viability Assay Kit (MilliporeSigma). To further test ANT MEF reliance on glycolysis, we treated cells cultured in Rho 0 media [DMEM/10% BGS/1 mM sodium pyruvate and uridine (50 μg/ml)] containing 40 mM 2-deoxyglucose (Sigma-Aldrich). Cells were visually inspected 12 hours later for survival.

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