Xf modified dmem

Lung cells were prepared as described below. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF^e96 Extracellular Flux Analyser and the XF cell mitochondrial stress test kit assay according to manufacturer’s instructions (Seahorse Biosciences, Billerica, MA). 3 x 10⁵ lung cells, prepared as described below, were resuspended in modified DMEM (XF modified DMEM, Seahorse Biosciences) (pH 7.4) supplemented with 1 mM pyruvate and 11 mM glucose for the mitochondrial stress test (mitochondrial function). Following basal energetic or respiration measurements, cells were sequentially treated with 1 μM oligomycin (ATP synthase inhibitor), 0.5 μM carbonylcyanide ptrifluoromethoxyphenylhydrazone (FCCP, proton ionophore), 1 μM antimycin A (complex III inhibitor) and 1 μM rotenone (complex I inhibitor). Mitochondrial function parameters of basal respiration (difference in OCR before and after antimycin A/rotenone treatment), oxidative ATP turnover (difference between OCR following oligomycin and before any treatment), maximal respiratory capacity (difference between OCR following treatment with FCCP and antimycin A/rotenone) and spare respiratory capacity (difference between OCR following treatment with FCCP and prior to any treatment) were determined.

The OCR was measured using an XF96 analyser (Seahorse Bioscience, North Billerica, MA, USA). Cells were seeded in 96-well XF96 cell culture plates at a density of 20,000 cells/well. After incubation for 48 h, the cells were treated with C118P (0.025, 0.05, 0.1 μM). The media were then removed, and the wells were washed in XF-modified DMEM (Seahorse Bioscience) at pH 7.4 supplemented with 1 mM glutamine (glycolysis and mitochondrial stress tests), 2.5 mM glucose, 1 mM sodium pyruvate, 0.5 mM carnitine, and 1 mM palmitate in complex with 0.2 mM BSA (mitochondrial stress tests) and incubated for 1 h at 37 °C without CO₂. The OCR was measured in the basal state (1 mM palmitate in complex with 0.2 mM BSA) or after the injection of 5 μM oligomycin, 1 μM 2-[2-[4-(trifluoromethoxy) phenyl] hydrazinylidene]-propanedinitrile (FCCP), and rotenone with antimycin A (both at 0.5 μM). After the Seahorse Bioscience experiments, the proteins were quantified to normalise the results.