Metabolic pathway analysis

LN229-TS cells were treated with PBS, TMZ (2 μM), 56MESS (1.1 μM) or NP56MESS (0.6 μM) for 36 h. Three samples, 5 million cells per sample, from each treatment group were collected. One-hundred microlitres of H₂O was added to each cell pellet to resuspend the cells followed by addition of 180 μl of methanol and 120 μl of chloroform. Samples were vortexed for 1 min and incubated at room temperature for 5 min. Afterwards, 150 μl of H₂O was added, followed by vortexing for 1 min and incubation at room temperature for 5 min and centrifugation at 10,000g for 10 min. Three-hundred-and-fifty microlitres of supernatant was collected and spun in a vacuum centrifuge concentrator at 225 g at room temperature for 5 h. Forty microlitres of 20 mM of ammonium acetate in H₂O was added for resuspension. MALDI-TOF MS and MetabolAnalyst were used for metabolic pathway analysis⁷². Metabolites were identified when the fold change was greater than or equal to 2 and q value less than or equal to 0.05.