Effects of Compound 1 on extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). (A) Change in ECARand OCR of Snu398 (top) and HepG2 (bottom) cells upon addition of lactate dehydrogenase A (LDHA) inhibitor. ECAR and OCR values were assessed by Seahorse XF Analyzer before and after addition of Compound 1, and changes observed 92 minutes after inhibitor addition were plotted as a function of inhibitor concentration. Results are means ± standard error (SE) of at least three independent experiments containing four wells per condition. EC50 values are means ± SE of at least three independent experiments. (B) Time-dependent changes in OCR in Snu398 (top) and HepG2 (bottom) cells in response to Compound 1 addition followed by 1 μg/mL oligomycin addition normalized to basal values. (C) Oligomycin-dependent OCR (OCROLG) of HepG2 cells as a function of Compound 1 concentration. OCROLG is defined as the OCR change to oligomycin following Compound 1 addition. Cells were measured in 5 mM glucose/0.5 mM glutamine or 0.5 mM glutamine DMEM. Values are presented as a percentage of the basal OCR. (D) OCR of HepG2 cells in response to Compound 1 in DMEM containing 5 mM glucose and 0.5 mM glutamine (left) or 0.5 mM glutamine alone (right). Oligomycin (1 μg/mL) and Compound 1 were added simultaneously after 14 minutes, and the time-dependent OCR was recorded. Residual OCR after oligomycin addition represents the non-mitochondrial and mitochondrial proton leak contributions to the total OCR. Non-mitochondrial OCR as determined by antimycin addition was unchanged after Compound 1 addition (data not shown). (E) OCR and ECAR responses of HepG2 mitochondria to Compound 1. Cells were permeabilized using 2 nM PMP in MAS containing 10 mM pyruvate/10 mM malate/4 mM ADP. For (B-E), each point represents means ± SD. *P <0.05, **P <0.01, ***P <0.001 compared to untreated controls.