Mitochondrial respiration measurements.

The oxygen consumption rate (OCR) was determined, as previously described (34, 35), in real time using the XFp extracellular flux analyzer (Seahorse Bioscience), which allows the measurement of many relevant parameters (total respiration, basal mitochondrial respiration, ATP-linked respiration, proton leak, maximal respiration, and spare respiratory capacity). Cells were seeded in an XFp cell culture miniplate at a concentration of 8,000 to 20,000 cells per well in their specified growth media. The next day, cells were washed twice with 180 μl of XF base medium (Seahorse Bioscience), followed by a 15-min incubation in the same medium. Cells were analyzed using the Seahorse XFp Cell Mito stress test kit according to the manufacturer's instructions. Briefly, after baseline measurement, the following injections were made: 1 μM oligomycin, 0.5 μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A.