For GC–MS profiling analysis of intracellular metabolites, cells were seeded and cultured in 6-well dishes (to about 1 million cells / 0.5 mg cell protein per well). Cells were washed quickly three times with cold PBS, and 0.45 ml cold methanol (50% v/v in water with 20 μM L-norvaline as internal standard) was added to each well. Culture plates were transferred to dry ice for 30 min. After thawing on ice, the methanol extract was transferred to a microcentrifuge tube. Chloroform (0.225 ml) was then added, the tube was vortexed and centrifuged at 10,000g for 5 min at 4 °C. The upper layer was transferred to another microcentrifuge tube, dried in a centrifugal evaporator and derivatized with 30 μl O-isobutylhydroxylamine hydrochloride (20 mg ml−1 in pyridine, TCI) for 20 min at 80 °C, followed by 30 μl N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (Sigma) for 60 min at 80 °C. After cooling overnight at 4 °C, the mixture was then transferred to SHIMADZU GCMS-QP2010 Plus for analysis.
