The Mt mouse line was generated at the ROSA26 locus in a C57BL/6J (The Jackson Laboratory) background using standard knock-in methodology. The targeting vector contained a CAG promoter, a loxP-flanked STOP cassette (STOP codons with all three reading frames), and a mitochondrial-targeted GFP2 (Mt-GFP) and nuclearly targeted tagBFP2 transgenes (nls-BFP) separated by T2A peptide sequences (37). A Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3′ end was included to enhance mRNA stability. The Nls-BFP signal was not used in this screen. A similarly engineered ROSA26 knock-in (Ai14 strain; 007908, The Jackson Laboratory), which contains a Cre-dependent cassette for the expression of cytosolic tdTomato (Cyto-tdTomato) was a gift from D. Page (The Scripps Research Institute, Florida). Mt mice were used in the primary screen. Progeny of Mt mice crossed with Ai14 mice that carried both fluorophores were used in the rescreen.

For respiration experiments using isolated mitochondria, C57BL/6J mice were grouped to receive dyclonine (25 mg/kg)–supplemented or standard water for 7 months starting at 2 months of age. The water supply was refreshed weekly. Mice were housed on a 12-hour light/12-hour dark cycle with ad libitum access to food and water. All experiments were performed during the light part of the diurnal cycle. Housing, animal care, and experimental procedures were consistent with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.

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