Measurement of cellular oxygen consumption rates.

The oxygen consumption rate (OCR) was determined using a Seahorse Bioscience XF24 Extracellular Flux analyzer at 37°C. AC16 cells were plated on XF24 microplates (Seahorse Bioscience) at 5.0 × 104 cells/well in DMEM media supplemented with 10% FBS and kept at 37°C in a 5% CO₂ humidified atmosphere 24 hours before SPTLC1 and SPTLC2 overexpression. Intact cellular respiration was assayed at posttransfection 24 or 48 hours under basal conditions (10 mM D-glucose, 10 mM pyruvate, 0% serum) and after the administration of various drugs as following: mitochondrial inhibitor oligomycin (oligo; 1 μM), mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM), respiratory chain inhibitor antimycin A (AA; 1 μM), and rotenone (ROT; 1μM). The XF24 microplate was loaded into the Seahorse XF24 analyzer following the manufacturer’s instructions.

Respiratory parameters were quantified by subtracting respiration rates at times before and after addition of electron transport chain inhibitors according to Seahorse Biosciences: basal respiration, baseline respiration minus AA-dependent respiration; ATP turnover, baseline respiration minus oligo-dependent respiration; H⁺ leak, oligo-dependent respiration minus AA-dependent respiration; and respiratory capacity, FCCP-dependent respiration minus AA-dependent respiration. Values were calculated for each individual well and averaged for each condition.