Myxothiazol

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), myxothiazol, antimycin A, rotenone, 2-deoxyglucose, oxamate, aminooxyacetate, glucose, sodium pyruvate and sodium palmitate were obtained from Sigma (St. Louis, MO, USA). L-glutamine was obtained from Invitrogen (Carlsbad, CA). Oligomycin was obtained from EMD (San Diego, CA, USA). Bovine Serum Albumin fraction V (fatty acid ultra-free) was obtained from Roche Diagnostics (Indianapolis, IN, USA). All compounds and medium were prepared according to the manufacturers' instructions unless indicated otherwise.

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.

Ubiquinone-1, stigmatellin, myxothiazol, and ascochlorin were purchased from Sigma-Aldrich (St. Louis, MO). AC0-12 was synthesized according to the method of 1-hydroxy-2-alkyl-4(1 H) quinolone synthesis^{36 (link)}. The MagicMedia™ E. coli Expression Medium was from Life Technologies (Carlsbad, CA). The Ultra Yield Flask™ was from Thomson Instrument Company (Oceanside, CA). The HisTrap HP column was from GE Healthcare (Buckinghamshire, England). The Econo-Pac 10DG column was from Bio-Rad (Hercules, CA). All of the other chemicals were of reagent grade and were obtained from commercial sources.

AA, antimycin A, Cf, rotenone, myxothiazol, Ry, RR, LaCl₃, EGTA, TTFA, TMPD, KCN and the remaining chemicals were from Sigma-Aldrich (Milan, Italy). CsA was from Novartis (Bern, Switzerland). FK506 was obtained from Calbiochem (San Diego, CA, USA). MitoSOX red, Rhod 2-AM, MitoTracker Red CMXRos and calcein AM were from Molecular Probes (Leiden, The Netherlands).

Bacterial lipopolysaccharide (LPS), iodoacetic acid (IAA), 2-deoxyglucose (2DG), indomethacin, 2-thenoyltrifluoroacetone (TTFA), oligomycin, rotenone, antimycin A and myxothiazol were from Sigma Aldrich (St. Louis, MO, USA). GKT137831, zileuton, PD146176, idebenone, piericidin A, trifluoromethoxy carbonylcyanide phenylhydroazone (FCCP), S-ethyl isothiourea (SEITU) and 1400W were from Cayman Chemical (Ann Arbor, MI, USA). GSK2795039 was from MedChem Express (Monmouth Junction, NJ, USA). All other chemicals were from Sigma Aldrich.

S1QEL1.1, S1QEL2.1 [5 (link)], S1QEL1.719 [25 ] and S3QEL3 [6 (link)] were provided by Calico Life Sciences LLC (South San Francisco, CA) and AbbVie Inc. (Chicago, IL). They were maintained as 10 mM stocks in dimethylsulfoxide at room temperature, shielded from bright light and diluted in dimethylsulfoxide as required before use. Amplex UltraRed (Cat No. A36006) was from ThermoFisher; atpenin A5 (Cat No. 11898) and FCCP (Cat No. 15218) from Cayman Chemicals; piericidin A (Cat No. 2738-64-9) from Santa Cruz Biotechnology; and horseradish peroxidase (HRP) (Cat No. P8125), superoxide dismutase 1 (SOD1) (Cat No. S7571), rotenone (Cat No. R8875), myxothiazol (Cat No. T5580), succinate (Cat No. 14160), glycerol 3-phosphate (Cat No. 94124), malonate (Cat No. 792535) and glutamate (Cat No. 1294976) were from Sigma.

Oxylipins used in this study (Supplementary Table 1) were obtained as previously described. 9-LOX and 13-LOX derivatives (9-HOT, 9-KOT, 13-HOT, and 13-KOT) were produced as in Prost et al. (2005 (link)); α-DOX derivative 2-HOT was synthesized as in Hamberg et al. (1999 (link)); non-enzymatically produced 10-HOT, 12-HOT, and the mixture 15-HOT/16-HOT, were obtained by singlet oxygen oxygenation (Przybyla et al., 2008 (link)). Oxylipin stocks were prepared in 95% ethanol and diluted in buffer or culture media to reach appropriate concentrations. OPDA and JA were purchased from Larodan Fine Chemicals. Additional products used for phenotype analysis and respiration assays were purchased from Sigma Aldrich with at least 95% purity: Antimycin A (A8674), Myxothiazol (T5580), Rotenone (R8875), Carboxin (45371), Sodium malonate (63409), KCN (60178), Oligomycin A (75351), Sodium ascorbate (A7631), tert-butyl hydroperoxyde (458139), 2(E)-hexenal (132659), Paraquat (36541), NADH (10107735001), ATP (A1852), ADP (01905), Sodium succinate (S2378), Dithiothreitol (D0632) and n-Propyl gallate (P3130).