Antimycin a

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in XF base medium (Seahorse Bioscience; Agilent, Santa Clara, USA) containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate (all from Merck), under basal conditions and in response to 1 μM oligomycin (Seahorse Bioscience) to block ATP synthesis, 0.25 μM FCCP (Seahorse Bioscience) to uncouple ATP synthesis from the electron transport chain, and 0.5 μM rotenone and antimycin A (Seahorse Bioscience) to block complex I and III of the electron transport chain, on an XF-96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s recommendations.

Oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, and antimycin A were obtained from Seahorse Bioscience (North Billerica, MA, USA) in XF Cell Mito Stress Test kit (#103015-100). Water soluble tetrazolium salts for the MTT assay were from Kishida Chemical Co., Ltd. (Osaka, Japan). 2′,7′-Dichlorofluorescein diacetate (D6883) and tert-butyl hydroperoxide (t-BHP; 458139) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Oxygen consumption rate (OCR) and extracellular media acidification rate (ECAR) were measured using a XF24 extracellular flux analyzer (Seahorse Bioscience), as described [48] (link). Cells were attached to XF24 plates using Cell-Tak (BDBioscience). OCR and ECAR were measured in unbuffered RPMI (Sigma) supplemented with 10 mM D-glucose (Sigma), 10 mM L-glutamine, and/or 10 mM sodium pyruvate, as indicated in the figure legends. Where indicated cells were treated with 0.09% dimethyl sulfoxide (DMSO) (Sigma), 1 µM oligomycin, 0.5 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 1.5 µM antimycin A, 0.75 µM rotenone (all seahorse bioscience). OCR and ECAR values were normalized to live cell number.

OCR was measured using a Seahorse Biosciences XF^e96 extracellular flux analyzer. Ten thousand cells per well were seeded in XF96 cell culture plates coated with poly-D-lysine (Sigma-Aldrich). Attachment of the cells was monitored after 1 h and 3 h, and cells were incubated over night at 37 °C with 5% CO₂. Before the assay, cells were washed twice with XF base media (unbuffered DMEM supplemented with 2 mM L-Glutamine [Sigma-Aldrich], 11 mM Glucose [Carl Roth], 1 mM Sodium Pyruvate [Sigma-Aldrich] at pH 7.4) and equilibrated for 1 h at 37 °C without CO₂. The OCR was measured afterwards using the following inhibitors: 2 μM oligomycin (Seahorse Biosciences), 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, Seahorse Biosciences), and 2 μM rotenone with 2 μM antimycin A (Seahorse Biosciences). For each condition (basal and after each inhibitor injection), cycles were performed in triplicate with 3 min mixing followed by 3 min measurement. After completion of the assay, the protein content per well was determined using the Bradford assay (Roti-Quant; Carl Roth). Absorption was measured at 595 nm using a PerkinElmer 2030 Explorer plate reader. The OCR was normalized to the protein content for each well, and the results of inner wells of the 96-well plate with the same cell density were evaluated for the analysis of the cell lines.