Ant1−/− (Slc25a4) Ant2-LoxP (fl) (Slc25a5) mice and Ant4−/− (Slc25a31) mice were described previously (16, 17). To generate liver-specific deletion Ant2fl/fl in mice, we crossed in an albumin-Cre transgenic line (the Jackson Laboratory, 003574). To delete the CypD gene product, we used mice lacking a functional Ppif gene (27). Ppif−/− mice were crossed to contain the Ant1−/− Ant4−/− and Ant2fl/fl-Alb-Cre alleles to generate quadruple gene-deleted mice. Ant-triple-null liver was generated in Ant1−/− Ant4−/− Ant2fl/fl-Alb-Cre mice, which were compared with Ant1−/− Ant4−/− and Ant2fl/fl littermates that lacked the albumin-Cre transgene, and hence, these mice only expressed ANT2 (ANT2-only), which is the predominant ANT family member normally expressed in liver. However, liver mitochondria from ANT2-only mice showed identical mitochondrial CRC and Ca2+-induced swelling to fully WT mitochondria. Moreover, mitochondrial ultrastructure in liver from neither Ppif−/− nor Ant1−/− Ant4−/− Ant2fl/fl-Alb-Cre mice showed alterations compared with WT mitochondria (see fig. S3C).

All experimental procedures with animals were approved by the Institutional Animal Care and Use Committee of Cincinnati Children’s Medical Center, protocols IACUC 2015-0047 and 2016-0069. We have complied with the relevant ethical considerations for animal usage overseen by this committee. The number of mice used in this study reflects the minimum number needed to achieve statistical significance based on experience and previous power analysis. Blinding was performed for some experimental procedures with mice, although blinding was not possible in every instance. Both sexes of mice were used in equal ratios.

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