Standardized Seahorse Assay Protocol

The Seahorse assays were conducted following a standardized protocol [36 (link)]. The Seahorse XFe96 Extracellular Flow Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to measure the oxygen consumption. An XF96 Cell Culture microplate was used to culture the LS174T cells, with a seeding density of 2.5 × 104 cells in 80 μL medium. The plate was incubated overnight. On the next day, the cell culture media was replaced with Seahorse XF modified media. Oligomycin (1 μmol/L), FCCP (1 μmol/L), and a mixture of rotenone (1 μmol/L) and antimycin A (1 μmol/L) were sequentially added during the measurements. To identify whether the compounds were ETC complex V/ATP synthase inhibitors, an equal volume of DMSO or compounds dissolved in DMSO solution was used as a substitute for oligomycin. To assess the inhibitory effects of the compounds on ETC complex I or III, DMSO alone was used as a substitute for rotenone and antimycin A, or these compounds were dissolved in DMSO.