Carbonyl cyanide p trifluoromethoxyphenylhydrazone fccp

OCR measurements were performed using a Seahorse Biosciences XF96 Extracellular Flux Analyzer. Cells were seeded at 6,000/well in XF96 microplates (Seahorse Biosciences, Santa Clara, CA). After a 24-h incubation, the growth medium was replaced with XF Assay Medium (Seahorse Biosciences) supplemented with 25 mM glucose (Sigma-Aldrich, Darmstadt, Germany). OCR measurements were made over 5-min periods following a 3-min mix period. The following components were sequentially added to the cells: 1 µg/mL oligomycin (Sigma-Aldrich), 300 nM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; Sigma-Aldrich), and 2 µM rotenone (MP Biomedicals, Tokyo, Japan). The spare respiratory capacity and coupling efficiency were calculated according to the Seahorse Bioscience instructions and the basal OCR was normalized to the cell number.

A list of all reagents, chemicals or kits used in this study can be found in Table S1. Lipopolysaccharide (LPS) and high molecular weight Poly(I:C) (PIC) were purchased from InvivoGen. MitoTEMPO (MT), N-acetylcysteine (NAC), antimycin A (AA), rotenone (ROT), potassium cyanide, 2-deoxyglucose (2-DG), oligomycin (OM) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were acquired from Sigma-Aldrich while S3QEL-2 was purchased from Cedarlane. IL-1β, IL-6, IL-10, TNF-α, and CXCL10 ELISA kits were purchased from R&D Systems. The IFN-α/IFN-β 2-Plex Mouse ProcartaPlex^TM Luminex Panel kit used was from Invitrogen. Tetramethylrhodamine, methyl ester (TMRM), MitoSox Red and CellROX Orange probes were from ThermoFisher. Antibodies against Complex II (SDHB) was from Abcam while antibodies recognizing SOD2 was purchased from Cell Signalling Technology. Antibodies targeting IRF3, pIRF3 (Ser385), IRF7, pIRF7 (Ser477), GPX4, Iκbα, Complexes I (NDUFB8), III (UQCRC2) and IV (COX4) were purchased from ThermoFisher.

All the compounds, coming from our in-house MBC library,³² (link) were prepared with a stock concentration of 25 or 10 mM in DMSO. The final % of DMSO in cell culture was not higher than 0.1%. Bafilomycin A1 (Baf1) (Enzo Life Sciences – BML-1100–0100) and okadaic acid (OA) (Sigma – O9381) were also prepared in DMSO. Deferiprone (DFP) (Sigma − 379409) was dissolved in water. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma, C2920) was dissolved in ethanol.

Cellular oxygen consumption rate (OCR) was measured using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse, Agilent Technologies, CA, USA). A total of 20,000 control and GRN KD SH-SY5Y cells per well were plated in DMEM supplemented with 10% FBS and 1% P/S. Then, 24 h later, cell growing media was replaced by 25 mM glucose, 1 mM Pyruvate and 2 mM L-glutamine containing XF Base medium. Cells were incubated for 1 h in a CO₂-free incubator at 37  °C allowing temperature and pH equilibration before loading into the XF24 analyzer. Mitochondrial function was determined through sequential addition of 1 µM oligomycin (Sigma-Aldrich, MO, USA), 0.5 µM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; Sigma-Aldrich, MO, USA) and 1 µM antimycin (Sigma-Aldrich, MO, USA)/1 µM rotenone (Sigma-Aldrich, MO, USA). This sequential addition allowed us to determine the basal oxygen consumption, oxygen consumption-linked to ATP synthesis (ATP), maximal respiration and the cellular spare capacity.

We used an XF96 extracellular flux analyzer (Agilent Technologies, Santa Clara, CA) to determine the bioenergetic profile of intact cells. Day-4 cells were used to seed XF96 plates (50 × 10³ cells/well) and were allowed to recover for 24 h. Cells were then incubated in bicarbonate-free DMEM (Sigma-Aldrich) supplemented with 11 mM glucose, 2 mM l-glutamine, and 1 mM sodium pyruvate, in a CO₂-free incubator for 1 h. Oxygen consumption rate (OCR) was recorded, to assess mitochondrial respiratory activity and glycolytic activity. OCR was recorded in basal conditions, and the cells were then treated sequentially with 2 µg/mL oligomycin, and 3 µM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (both from Sigma-Aldrich). Non-mitochondrial respiration (OCR after treatment with 1 μg/mL antimycin A (Sigma-Aldrich)) was subtracted from all OCR measurements. ATP-linked respiration was estimated from the difference between the basal and oligomycin-inhibited respiration rates, and proton-leak respiration was obtained by subtracting non-mitochondrial respiration from the OCR measured after oligomycin treatment. Maximal respiratory capacity was determined as the rate of respiration in the presence of the uncoupler FCCP. Three independent replicates of each measurement were generated, and results were normalized according to cell concentration.