Glycolysis examination

The extracellular acidification rate (ECAR) of the cells was determined using a Seahorse XF Glycolysis Stress Test Kit with an XF96 Extracellular Flux Analyzer (Seahorse Bioscience; Agilent Technologies, Inc.) according to the manufacturer's instructions. Briefly, 2×10⁴ cells were plated into XF96 cell plates (Agilent Technologies, Inc.) and treated with 20 nM FK866 alone or in combination with 500 µM NMN at 37°C for 24 h. After washing with XF assay media, each well of the XF96 cartridge was sequentially injected with glucose (detection of glycolysis), oligomycin (an ATPase inhibitor, which restrains mitochondrial ATP production) and 2-DG (a glucose analog, which inhibits glycolysis). Glucose uptake, lactate production and ATP concentration were measured in cell lysates using a Glucose Uptake Colorimetric Assay Kit (BioVision, Inc.), Lactate Colorimetric/Fluorometric Assay Kit (BioVision, Inc.) and ATP Colorimetric/Fluorometric Assay Kit (BioVision, Inc), respectively. The enzymatic activities of PKM2 and LDHA were determined using a Pyruvate Kinase Activity Assay Kit (Beijing Solarbio Science & Technology Co., Ltd.) and Lactate Dehydrogenase Activity Colorimetric Assay Kit (BioVision, Inc.), respectively. All measurements were performed according to the manufacturer's protocols and normalized to the cell protein levels.