OCRs and ECARs were measured using an XF24 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA). HeLa cells (15×103) transfected with siCtrl or siOPA1 were plated on XF24 microplates 3 days before OCR measurements. Dual-analyte sensor cartridges were soaked in XF Calibrant Solution (Seahorse Biosciences) in 24-well cell-culture microplates overnight at 37°C to hydrate. Approximately 1 h prior to experimentation, injection ports on the sensor cartridge were filled with oligomycin (1 µM), FCCP (1 µM), and rotenone (1 µM) plus antimycin A (1 µM). Plates were then loaded into the XF24 instrument for calibration. For measuring oxygen consumption, the DMEM of HeLa cells was replaced by DMEM supplemented with NaCl (143 mM), Phenol Red (3 mg/ml), glucose (10 mM), glutamine (2 mM) and pyruvate (2 mM) at pH 7.4, and the plates were maintained at 37°C 1 h prior to experimentation. Plates were then loaded into the Seahorse XF24 analyser following the manufacturer's instructions.
ATP measurements in HeLa cells were determined using an ATP Colorimetric/Fluorometric Assay kit (Abcam). Intracellular ATP levels were determined using the colorimetric assay, following the manufacturer's instructions, on 1.106 HeLa cells transfected with control siRNA or OPA1 siRNA. ATP content was measured in duplicate (570 nm) and calculated per microgram of protein.
ATP measurements in HeLa cells were determined using an ATP Colorimetric/Fluorometric Assay kit (Abcam). Intracellular ATP levels were determined using the colorimetric assay, following the manufacturer's instructions, on 1.106 HeLa cells transfected with control siRNA or OPA1 siRNA. ATP content was measured in duplicate (570 nm) and calculated per microgram of protein.