Mitochondrial respiration analysis

Mitochondrial respiration was measured using a Seahorse Xfe24 Analyzer (Agilent Technologies). After 4 days of treatment, cells were seeded onto a XF24 microplate (30,000 cells/well) in normal culture medium and cultured for 16–18 h. The culture medium was then replaced by analysis medium (DMEM, high glucose, without FBS, and sodium bicarbonate) and incubated without CO₂ for 1 h before analysis according to the manufacturer’s protocol. Basal oxygen consumption rate (OCR) was measured first, then after sequential injections of three compounds that affect bioenergetics: 1 μM oligomycin (Sigma, St. Louis, MO, USA), 0.5 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma) and 2 μM rotenone (Sigma). After completion of the analysis, protein was measured using a BCA assay (Pierce Biotechnology). Results were normalized to the protein OD value of corresponding wells.