Metabolomic Analysis of Cultured Cells

For the LC-MS metabolomic analysis, the sample extraction was performed as described previously [81 (link),82 (link)]. Briefly, 5 × 10⁷ cells were used for each sample. Cells were rapidly cooled in a dry ice/ethanol bath to 4°C, centrifuged at 1,300g, 4°C for 10 minutes, washed with 1× PBS, and resuspended in extraction solvent (chloroform:methanol:water, 1:3:1 volume ratio). Following shaking for 1 hour at 4°C, samples were centrifuged at 16,000g at 4°C for 10 minutes, and the supernatant was collected and stored at −80°C. The analysis was performed using separation on 150 × 4.6 mm ZIC-pHILIC (Merck, Kenilworth, NJ) on Dionex UltiMate 3000 RSLC (Thermo Fisher Scientific Waltham, MA) followed by mass detection on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Waltham, MA) at Glasgow Polyomics.