Carcinoscope

For the metabolome analysis, U266/Ctrl and U266/MYC cell lines treated with CB-839 1μM for 48h were prepared in triplicates. The absolute concentration of 116 metabolites was measured using capillary electrophoresis mass spectrometry (CE-TOFMS and CE-QqQMS) in the cation and anion analysis modes for analyzing cationic and anionic metabolites, respectively by the metabolome analysis package Carcinoscope provided by Human Metabolome Technologies (HMT). Samples were prepared following HMT’s Sample Preparation Protocol. Briefly, (6 × 10⁶ cells/sample) was used for the extraction of intracellular metabolites. Cells were collected from 100mm plate and washed twice using washing solution (5% mannitol). The cells were then treated with 800 μL of methanol and vortex for 30 s in order to inactivate the enzymes. Next, the cell extract was treated with 550 μL of Milli-Q water containing internal standard (H3304-1002, Human Metabolome Technologies, Inc., Tsuruoka, Japan) and vortex for another 30 s. The extract was obtained and centrifuged at 2300 × g and 4°C for 5 min and then 350 μL of upper aqueous layer was centrifugally filtered through a pre-washed ULTRAFREE MC PLHCC centrifugal filter units (provided by HMT) at 9100 × g and 4°C for 90 min. Samples were evaporated under vacuum conditions at room temperature 1500 rpm, 1000 Pa, 2–3 h (until no liquid remains in the filter cup).