MIA PaCa-2 cancer cells stably expressing GFP or human MAGE-A6 (provided by K. Scott, Baylor College of Medicine) were cultured in Dulbecco’s modified Eagle’s medium high glucose, 10% FBS, and 2.5% horse serum. Cells were plated in a 96-well plate, 1 × 104 cells per well, and treated with 2 mM 2-DG glycolysis inhibitor (Sigma) for 12 days. Viability was assessed using alamarBlue viability reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were incubated with 10% reagent in complete medium for 4 hours, and increased fluorescence was detected using an excitation between 530 and 560 nm and an emission at 590 nm.

For nontargeted metabolomics analysis, MIA-PaCa-2 cells stably expressing GFP or MAGE-A6 were grown in standard media or 2 mM 2-DG for 2 to 10 days. Nontargeted metabolomics was performed by the NIH West Coast Metabolomics Center (n = 6 for each group) (47). Data were normalized and analyzed by MetaboAnalyst (48).

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