Measurement of mitochondrial oxygen consumption rate

The oxygen consumption rate (OCR) was measured by the extracellular flux analyzer XF24 (Seahorse Bioscience, Houston, TX, USA) as we previously described [16]. Cells were plated at 4 × 10⁵ cells/well in a Seahorse 24-well V7 microplate (Seahorse Bioscience) and cultured in complete DMEM growth medium for 24 h in a 5% CO₂ incubator at 37 °C. Then, the medium was removed and cells were incubated in XF assay medium in the absence of NaHCO₃ and FBS for 1 h at 37 °C in measuring chamber without CO₂ input. The mitochondrial complex inhibitors (oligomycin, FCCP, antimycin A1/ rotenone) were freshly prepared in XF assay media. After 26 min of measuring the basal respiration, oligomycin (2.5 μM) was injected, followed by FCCP (1 μM) at 50 min, and antimycin A (2.5 μM) /rotenone (2.5 μM) at 74 min. OCR was recorded as pMoles per minute. ATP turnover was measured after the treatment with oligomycin. Averages of three wells were taken per data point.