Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR)

Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent Technologies, USA) was used to measure cellular glycolytic capacity and mitochondrial function, following the manufacturer’s instructions of Seahorse XF Glycolysis Stress Test Kit or Seahorse XF Cell Mito Stress Test Kit. Cells were plated in XF96 Cell Culture Microplates (6×10³ cells/well) and cultured overnight. Then, the cells were treated with NG52 for 12 hours prior to the measurement. For ECAR analysis, 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxy-glucose were added automatically. For OCR analysis, 1.5 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone/antimycin A were added automatically. Seahorse XF-96 wave software was used to calculate the data.