Mitochondrial Stress Analysis in MEFs

The XF Cell Mito Stress Test was performed as previously described [18 (link)]. Briefly, forty thousand E1A/RAS MEFs were plated in Seahorse 96-well cell culture microplates coated with Cell-Tak (Corning^™) and subjected to the Seahorse XF Cell Mito Stress Test, according to manufacturer’s protocol. Briefly, cell medium was replaced with Seahorse XF Base Medium (supplemented with 100 mM Pyruvate, 200 mM Glutamine, and 2.5 M Glucose) and incubated in a non-CO₂ incubator for one hour before the start of the assay. Basal OCR was measured using the Seahorse XFe96 Extracellular Flux analyzer. Measurements were performed after injection of three compounds affecting bioenergetics: 1 μM oligomycin (Seahorse Bioscience, North Billerica, MA, USA), 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Seahorse Bioscience) and 0.5 μM Rotenone/Antimycin A (Seahorse Bioscience). Data are representative of three biological replicates.