Measuring Mitochondrial Respiration in hESCs

For mitochondrial respiration measurements, stable cell lines with lentiviral PATZ1 knockdown were used. The oxygen-consumption rate (OCR) and extracellular acidification rate (ECAR) of H1 hESCs were measured by Seahorse XF Extracellular Flux Analyzer (Agilent) and was performed according to the manufacturer's protocol. Briefly, 20,000 hESCs/well were seeded onto a Matrigel^®-coated XF96 cell culture microplate (Agilent) and cultured overnight in 80 μl of hESC culture medium. One hour before the assay culture medium was changed to pH 7.4 XF assay medium supplemented with 1 mM pyruvate (Gibco), 2 mM glutamine (Gibco), and 10 mM glucose (Sigma), and incubated at the incubator without supplied CO₂ for 1 h before the completion of probe cartridge calibration. Basal respiration was measured in the XF assay medium without oligomycin, and mitochondrial function was measured by injecting 2 μM oligomycin (Sigma), 2 μM FCCP (Sigma), and 1 μM rotenone (Sigma) mix with 1 μM antimycin A (Sigma) for OCR assays and 10 mM glucose, 1 mM oligomycin, and 50 mM 2-DG (Sigma) for ECAR assays. After the test, the total protein in each well was measured by SRB (Sigma) method and the data were normalized on proteins. OCAR and ECAR results were analyzed using the calculation method described by Mookerjee et al. 43 (link), 44 (link). Equations were available in the Supplementary Table 6.