Mid49

After treatments, murine heart tissues and primary cardiomyocytes were collected, digested and analyzed using a BCA assay (for protein concentration, Thermo Fisher Scientific, USA). Protein samples of each group were separated using SDS-PAGE gels, transferred to the polyvinylidene difluoride membrane (PVDF, Millipore, USA), and then incubated overnight at 4 °C with specific antibodies against Mst1 (1:1,000, Cell Signaling Technology); t-Drp1 (1:1,000, Millipore, USA); p-Drp1^S616 (1:500, Cell Signaling Technology, USA); p-Drp1^S637 (1:500, Cell Signaling Technology, USA); Mff (1:1,000, Proteintech, USA); Mid49 (1:500, Proteintech, USA); Mid51 (1:1,000, Proteintech, USA); Fis1 (1:1,000, Proteintech, USA); Mfn1 (1:500, Proteintech, USA); Mfn2 (1:5,000, Abcam, USA); Opa1 (1:2,000, Abcam, USA); caspase-3 (1:1,000, Cell Signaling Technology, USA); cleaved caspase-3 (1:1,000, Cell Signaling Technology, USA) and β-actin (loading control, 1:1,000, Cell Signaling Technology). Then, blots were incubated with horseradish peroxidase (HSP)-conjugated secondary antibody (1:5,000, Proteintech, USA) for 1 h. Finally, blots were scanned and detected by the luminescence method. Band intensity was analyzed using the Image J software (Version 1.45).

Apogossypol and Leflunomide from ApexBio (Boston, MA, USA), A-1331852, A-1210477, ABT-199, Z-VAD.FMK and CCCP from Selleck (Houston, TX, USA), Teriflunomide, norhydroguaiaretic acid (NDGA), Ivermectin, Terfenadine, Suloctidil, orotate and uridine from Sigma Aldrich (St Louis, MO, USA), MitoTracker Deep Red FM from Thermo Fisher (Loughborough, UK) were used. Antibodies against BAP31, RTN4, BiP, PDI, CHOP, DHODH and tubulin from Abcam (Cambridge, UK), CLIMP-63 and BAX (6A7) from Enzo Life Sciences (Exeter, UK), TIM22 and KNT-1 from Sigma, HSP60, Cytochrome c, BAX, OPA1 and DRP-1 from BD Biosciences (San Jose, CA, USA), phospho-DRP-1 (S616), phospho-DRP-1 (S637), MFN1 and MFN2 from Cell Signaling Technologies (Danvers, MA, USA), BAK (AB-1) from Calbiochem (Watford, UK), MFF, MID49 and MID51 from ProteinTech (Manchester, UK) and GAPDH from Santa Cruz Biotechnologies (Santa Cruz, CA, USA) were used. For RNA interference, cells were transfected with 10 nM of siRNAs against DHODH (SI00363384 and SI00363391) purchased from Qiagen Ltd. (Manchester, UK), using Interferin (Polyplus Transfection Inc, NY), according to the manufacturer’s protocol and processed 72 h after transfection.

Cell extract preparation was performed as described previously (Bienholz et al. 2017 (link)). The shown proteins were visualised using a rabbit antibody against Phospho-Drp1-S637 (Cell Signaling, Danvers, Massachusetts, USA, #4867), Phospho-Drp1-S616 (Cell Signaling, #4494), Drp1 (Cell Signaling, #5391), Opa1 (Cell Signaling, #80471), Fis1 (Thermo Scientific, Waltham, Massachusetts, USA, #PA5-22142), Mfn1 (Merck, Darmstadt, Germany, #ABC41), Caspase-3 (Cell Signaling, #9662), Mff (Cell Signaling, #84580), Phospho-Mff (Cell Signaling, #49281), MiD51 (Proteintech, Rosemont, Illinois, USA, 20164-1-AP), MiD49 (Proteintech, 28718-1-AP) and a mouse antibody against Oma1 (Santa Cruz, Dallas, Texas, USA, sc-515788) and Mfn2 (Abcam, Cambridge, United Kingdom, #ab56889), all in 1:1000 dilution. Anti-rabbit IgG (Sigma-Aldrich, St. Louis, Missouri, USA, #A8275) and anti-mouse IgG (Sigma-Aldrich, #A4416) conjugated with peroxidase was used as secondary antibody in 1:5000 dilution. The chemiluminescence signals (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were recorded using the gel documentation system Fusion Pulse 6 (Vilber Lourmat, Eberhardzell, Germany) and quantified by the software Bio1D (Vilber Lourmat, Eberhardzell, Germany). Gels stained with Coomassie were used as a loading control.