Murine Cell Culture and Cytotoxicity Assays

RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium High Glucose (4.5 g/L) and NaHCO₃ (1.5 g/L) (DHG-L1, Gibco-Life Technologies, Carlsbad, CA, USA), supplemented with 10 % heat-inactivated foetal bovine serum (FBS, Gibco). HeLa cells (ATCC) were cultured in Minimum Essential Medium (MEM, Gibco) supplemented with nonessential amino acids (Gibco), 1 mM pyruvate (Gibco) and 10% FBS. DRP1^+/+ and DRP1^−/− MEFs (a generous gift from Prof. Ishihara, Kurume University, Japan) were kept in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10 % FBS and EB1 and rho⁰ HeLa cells (kind gift from Prof. Hayashi, University of Tsukuba, Japan) in DMEM high glucose (DHG, Gibco) supplemented with 1 mM pyruvate, 50 µg/ml uridine (Sigma-Aldrich, St. Louis, MO, USA) and 10% FBS. BMDMs were obtained from femurs and tibias of 6-to-8-week-old C57BL/6 mice, as previously described^{94 (link)}. Cells were seeded and treated in culture plates (Corning-Costar, Lowell, MA, USA).
When indicated, myxothiazol (10 nM, Sigma-Aldrich), antimycin A (100 nM, Sigma-Aldrich), Mito-TEMPO (500 µM, Sigma-Aldrich), ruthenium red (1 or 10 µM, Sigma-Aldrich), tunicamycin (10 µM, Sigma-Aldrich), TNFα (10 ng/ml, Sigma-Aldrich) or cycloheximide (10 µg/ml, Sigma-Aldrich) were added to the culture media for the indicated times.