Lethal cancer cells buddy up to survive naturecancer
. KPC7940B cells were a gift from G. Beatty . KPC-MT3 cells were a gift from D. Tuveson . PATC53 cells were obtained from ATCC. Cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum and routinely tested for mycoplasma contamination using MycoAlert Plus . DMSO, oligomycin, phenformin and aminooxyacetic acid were obtained from Sigma,-ISRIB was obtained from Cayman Chemical and GCN2iB was obtained from MedChemExpress. IACS-10759 was obtained from Selleck Chem.
In both acquisition modes, key parameters of Agilent Jet Stream ESI were a gas temperature of 275 °C, gas flow of 14 liters minand capillary voltage of 3,000 V. For negative-mode MS, delta EMV was 350 V, cycle time was 500 ms and the cell accelerator voltage was 4 V. For positive acquisition mode MS, the delta EMV was set at 200 V with no change in cycle time and cell accelerator voltage.
For separation, an Agilent ZORBAX RRHD Extend C18 column and ZORBAX Extend Fast Guards for UHPLC were used. The LC gradient profile was at 0.25 ml min, 0–2.5 min, 100% A; 7.5 min, 80% A and 20% C; 13 min 55% A and 45% C; 20 min 1% A and 99% C; 24 min 1% A and 99% C; 24.05 min 1% A and 99% D; 27 min 1% A and 99% D; at 0.8 ml min–1, 40 min, 100% A. The column temperature was kept at 35 °C, and samples were at 4 °C. The injection volume was 2 µl.
The MassHunter Metabolomics Dynamic MRM Database and Method was used for target identification. Key parameters of the Agilent Jet Stream ESI were gas temperature of 150 °C, gas flow of 13 liters min, capillary voltage of 2,000 V, nozzle at 500 V and detector delta EMV at 200 V.Chromatography followed the same method and setup as the 1290 Infinity II LC method listed above for the 6740 parameters. MS was performed with an Agilent G6545B MS Q-TOF with Dual AJD ESI sources in Profile Mode.
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