Mitochondrial respiration assay

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in PC3 cells seeded at 20,000 cells per well, treated with the indicated canagliflozin and radiotherapy doses, and analyzed 48 hours later using the Agilent Technologies Seahorse XFe96 extracellular flux analyzer system (Santa Clara, CA). Briefly, incubation medium was changed one hour before the start of the assay to Seahorse XF medium, which was supplemented with 25 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate. Different ETC complex inhibitors (oligomycin, FCCP, and rotenone/antimycin-A) were then sequentially added during the assay at pre-optimized concentrations of 1.5 μM, 1 μM, and 0.5 μM, respectively. Then, OCR, ECAR, basal respiration, maximal respiration, ATP production, non-mitochondrial respiration, spare respiratory capacity, OCR:ECAR ratio were calculated⁷³.

The following formulas were used to calculate mitochondrial function variables (basal respiration, maximum respiration, mito-ATP production, and basal ECAR:OCR); Basal respiration= final rate measurement before the initial injection–non-mitochondrial respiration rate. Maximal respiration= maximum rate measurement after FCCP injection–non-mitochondrial respiration. ATP production= final rate measurement before Olig. injection–minimum rate measurement after Olig. injection. The following formula was used to convert ATP production to mitochondrial ATP production rate: MitoATP production rate (pmol/ATP/min)= OCR_ATP (PMOL/O₂/min) x 2 (pmol O/pmol O₂) x P/O (pmol ATP/pmol O), where P/O was adjusted to a verified value of (2.75). Basal OCR:ECAR ratio=The OCR mean from the last three baseline data points, divided by the ECAR mean of the same last three baseline data points. The default buffer factor of 2.4 was taken into account and implemented prior to performing the calculations.