Oligomycin

OCR was measured in NRVMs using Seahorse XF24 Flux analyzer (Agilent Technologies) following the instructions of Seahorse XF Cell Mito Stress Test Kit with slight modifications as previously described (Wang et al. 2017 ).
Briefly, NRVMs or hESC derived cardiac myocytes were seeded into a XF24 cell culture microplate at 1 × 10⁵ cells per well. For TRIM72 overexpression or knockdown, cells were infected with TRIM72 adenovirus or transfected with TRIM72 siRNA for 48 h. Before the assay, cells were incubated in unbuffered medium (DMEM) (D5030, Sigma) supplemented with 32 mM NaCl, 25 mM glucose and 2.5 mM sodium pyruvate, pH 7.4) for 1 h at 37 °C without CO₂. Then basal respiration was assessed in untreated cells. ATP turnover was assessed after adding 1 µM Oligomycin (ab141829, Abcam). Maximum respiratory capacity was determined by adding 1 µM arbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Finally, nonmitochondrial respiration was measured after adding 1 µM Rotenone (R8875, Sigma) and 1 µM Antimycin A (ab141904, Abcam). Upon completion of the measurement, the OCR value (pMoles/min) was normalized with the total protein amount of each well and used for further analysis.

The SeaHorse Extracellular Flux Analyzer (SeaHorse Bioscience, United States) was used to analyze microglial metabolism as previously described (McIntosh et al., 2019 (link)). Microglia (50,000 cells/well; final volume 200 μl; 4–6 replicates/sample) were seeded on SeaHorse cell culture microplates, the sensor cartridge was hydrated by adding SeaHorse XF Calibrant solution (200 μl) to each well and samples were left overnight in a CO₂-free incubator at 37°C. Cells were washed, assay media added to give a final volume of 200 μl/well and incubation continued (37°C; 1 h; CO₂-free incubator). For the assay, oligomycin (20 μM; AbCam, United Kingdom), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (20 μM; FCCP; Sigma-Aldrich, United Kingdom) and antimycin A (40 μM; Sigma-Aldrich, United Kingdom) were loaded into the appropriate ports for sequential delivery at 24 min intervals and, following calibration, Oxygen Consumption Rate (OCR) was measured at 8 min intervals for a total of 96 min and automatically calculated using the SeaHorse XF96 software.

Irinotecan hydrochloride, 2-thenoyltrifluoroacetone (TTFA), hydroxyurea (HU), 2′,7′-dichlorofluorescin diacetate (DCFDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2-deoxy-d-glucose (2-DG), crystal violet, and antimycin A were purchased from Sigma Aldrich (Deisenhofen, Germany). Doxorubicin was purchased from Enzo Life Science (Lörrach, Germany). Chloramphenicol was purchased from Carl Roth GmbH (Karlsruhe, Germany). Rotenone, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and oligomycin were purchased from Abcam (Cambridge, UK). 3,3′-Dihexyloxacarbocyanine iodide (DiOC₆(3)) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). DMSO was used as a treatment control.