The mitochondrial respiration capabilities of LLC-PK1 SCR and PC2 KD cells were tested using the Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). Cells were plated in 96-well plates at a density of 8 × 104 cells per well 24 hours before the assay. Two hours before analysis, cells were washed twice with and then changed to Seahorse Assay Medium (Agilent Technologies) containing d-glucose (4.5 g/liter), 2 mM l-glutamine, and 1 mM sodium pyruvate before being placed in a CO2-free incubator at 37°C. Basal OCR measurements were taken before the sequential addition of 2 μM Oligomycin A, 2 μM FCCP, and 1 μM rotenone to measure ATP production and proton leak, maximal mitochondrial respiration, and nonmitochondrial respiration, respectively. After completion of Seahorse analysis, OCR results were normalized to cell number by lysing cells and quantifying the double-stranded DNA (dsDNA) in each well using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Experiments were performed in quadruplicates or greater.

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