Pemetrexed

Three human MPM cell lines (H28, H2452 and MSTO-211H) were obtained from ATCC (Manassas, VA) and cultured as previously described.^{26 (link)} Human primary DMPM cultures (MesoII and STO) were derived from tumour samples of patients who underwent surgery, and were maintained in DMEM/F12 (Gibco) under standard culture conditions for less than 20 passages.^{24 (link)} Cells were routinely tested for mycoplasma. Analyses of mitochondrial function and glycolysis of MSTO-211H and H2452 cells were performed with the Seahorse XFp Metabolic Flux Analyzer (Agilent Technologies, Santa Clara, CA), showing that these cells have both a normal aerobic metabolism and aerobic glycolysis, though to a different extent, as reported in the Supplemental Methods and Supplementary Fig. S1.
Co-transduction of MesoII cells with luciferase vectors and Firefly-luciferase (F-luc) activity assessment were performed according to previously established methods.^{23 (link),24 (link)} Pemetrexed and gemcitabine were gifts from Eli Lilly (Indianapolis, IN), while the LDH-A inhibitors NHI-2 and NHI-Glc-2 were synthesised as described previously.^{17 (link),27 (link)} Drugs were dissolved in sterile water (gemcitabine) or dimethyl sulfoxide (DMSO, Pemetrexed and LDH-A inhibitors) and diluted in culture medium immediately before use. In each experiment, we did not use concentrations higher than 0.1% DMSO.

Metformin hydrochloride and crystal violet were purchased from Sigma-Aldrich (St. Louis, MO, United States). Pemetrexed was obtained from Eli Lilly and Company (Indianapolis, IN, United States).

The lung adenocarcinoma cell lines CL1-0, CL1-5, and A549 were provided by Dr. Cheng-Wen Wu and Dr. Pan-Chyr Yang [44 (link),45 (link),46 (link)]. Cells were grown in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum and cultured at 37 °C in a humidified atmosphere with 5% CO₂. The folate-deficient medium, RPMI 1640 medium without folic acid, was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum was dialyzed at 4 °C for 16 h against 10 volumes of sterile phosphate-buffered saline and used under the FD condition to minimize the amount of folate from exogenous sources. Pemetrexed from Lilly (Indianapolis, IN, USA). Gefitinib (Iressa) was obtained from AstraZeneca (Macclesfield, Cheshire, UK), and erlotinib (Tarceva) was obtained from Roche (Basel, Switzerland). Antibodies specific for E-cadherin (H-108), vimentin (V9), SLUG (A-7) (Santa Cruz Biotechnology, Dallas, TX, USA), N-cadherin (22200002) (Novus International, Saint Charles, MO, USA), Sox9 (EPR14335) (Epitomics, Wembley, London, UK), and actin (AC-15) (Sigma-Aldrich, St. Louis, MO, USA) were used in Western blot analysis.