Milli q water

Culture medium in Φ10 cm dishes was aspirated and cells were washed twice with 5% mannitol solution (10 mL followed by 2 mL). Cells were treated with 800 μL of methanol and left for 30 seconds to inactivate enzymes. The cell extract was then treated with 550 μL of Milli-Q water containing internal standards (H3304-1002; Human Metabolome Technologies, Tsuruoka, Japan) and left for another 30 seconds. The extract was centrifuged (2300 × g at 4°C for 5 minutes) and then 800 μL of the upper aqueous layer was centrifugally filtered (9100 × g at 4°C for 120 minutes) through a Millipore 5-kDa cutoff filter (UltrafreeMC-PLHCC, Human Metabolome Technologies) to remove macromolecules. The filtrate was centrifugally concentrated and re-suspended in 50 μL of ultrapure water. The obtained samples were sent to Human Metabolome Technologies for analysis by capillary electrophoresis time-of-flight mass spectrometry.

Blood samples of 3 ml from fasting participants were acquired at the outpatient clinic of our department between 0800 and 0900 and stored at −80°C. A plasma volume of 50 µl was added to 450 µl of methanol containing internal standards and mixed. Then, 500 µl of chloroform and 200 µl of MilliQ water were added, and the resulting solution was mixed again. After centrifugation at 2300g for 5 min, an aliquot of 400 µl from the aqueous layer was sampled and filtered using a 5 kDa membrane filter, followed by drying under reduced pressure. The residue was reconstituted with 25 µl MilliQ water for CE-TOFMS analysis (all reagents from Human Metabolome Technologies Inc., Tsuruoka, Japan).

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).