D glucose solution

Cantharidin (purity≥98%) was purchased from Chengdu Must Bio-Technology Co., Ltd (Chengdu, China). Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, and penicillin/streptomycin were from Gibco (Gibco, Grand Island, NY, USA). Primary antibodies to PKM2, GLUT1, and MCT1 were obtained from Abcam (Abcam, Cambridge, UK). Antibody against EGFR, PIN1, importin α5, and LDHA were purchased from CST (Cell Signaling Technology, Danvers, MA, USA), MCT4 from Proteintech, and β-actin and PKM from ABclonal (ABclonal, Woburn, MA, USA). D-(+)-Glucose solution, sodium pyruvate solution, and L-glutamine were provided by Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Other standard substances were obtained from Yuanye Biotechnology Co., Ltd (Shanghai, China). Glycolysis Stress Test Kit and Cell Mito Stress Test Kit were purchased from Seahorse Biosciences (Seahorse Biosciences, North Billerica, MA, USA).

For glucose deprivation and hypoxia experiments, cells were equally seeded in 75 cm² cellstar filter top plastic flasks and kept in the incubator overnight to ensure adherent growth. Expansion and treatment of the cells for starvation experiments was performed in DMEM medium (DMEM Life Technologies, Paisley, UK) containing 1 mM pyruvate (Sigma). For glucose deprivation experiments glucose- and serum-free medium (DMEM Life Technologies) was added to equally subconfluent cells. In the case of low glucose concentration, 5 mM of sterile D-(+)-glucose solution (45%, Sigma Aldrich, St. Louis, USA) was applied. For hypoxia experiments, cells were kept in serum-free medium and transferred into a hypoxia chamber (Binder incubator, Tuttlingen, Germany). The required oxygen concentration (0.1%, 5% and 21% O₂) was set before the particular experimental condition. For amino acid starvation, DMEM medium lacking L-arginine, L-glutamine and L-lysine (DMEM Life Technologies) supplemented with 1 mM pyruvate (Sigma) was used. To generate the 20% amino acid condition, the appropriate amino acids (L-arginine hydrochloride and L-lysine hydrochloride from Sigma-Aldrich, St Louis, MO, USA and L-glutamine from Gibco, Invitrogen, Carlsbad, CA, USA) were added in 0.2 fold concentration of the control condition.

Monoclonal antibody was transiently expressed in the Expi293 system (ThermoFisher, A14635). In brief, antibody HC and LC plasmids were co-transfected at a ratio of 1:2.5 with transfection reagent FectoPRO (Polyplus 116-010). After 24 h of transfection, 300 mM of sterile sodium valproic acid solution (Sigma-Aldrich, P4543) and 45% D-(+)- glucose solution (Sigma Aldrich, G8769-100ML) were added to feed cells. After 4–5 days of transfection, supernatants were collected, sterile-filtered (0.22 μm), and IgG was purified with Protein A sepharose beads (GE Healthcare 17-5280-04).

Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed at 10 weeks of dietary intervention before surgery and at 13 weeks (10 weeks of diets + 3 weeks after surgery and plasmid injection). Before both tests, mice underwent overnight fasting and body weight measurement. Peripheral blood samples were collected from the tail vein after dissection by a sterile scalpel; glucose level was assayed by a glucometer. For the GTT, 2 g/kg of glucose (10% D-glucose solution, Sigma-Aldrich, Burlington, MA, USA) was injected intraperitoneally. Glucose levels were measured before injection and at 15, 30, 45, 60, 90 and 120 min after injection. For ITT, 0.5 IU/kg of human insulin (Humulin, Lilly France GmbH, Paris, France) was injected intraperitoneally. Glucose levels were measured before injection and at 15, 30, 45, 60, 90, 120, 150 and 180 min after injection.

The GC cell line 58As9 was established and kindly provided by Dr. K. Yanagihara (National Cancer Center, Chiba, Japan)^{32 (link)}. This cells line was further authenticated by the JCRB Cell Bank (Osaka, Japan). MKN74 cells were purchase from Cell Bank, RIKEN Bio Resource Center (Tsukuba, Japan). HIF-1α knockdown (KD) cells were established by transfecting plasmids harboring HIF-1α RNAi sequences into 58As9 and MKN74 cells, as previously described^{11 (link),16} . The cells were cultured at 37 °C in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-iNACtivated fetal bovine serum and 100 μg/mL kanamycin (Meiji, Tokyo, Japan). Cells were cultured under either normoxic (20% O₂ and 5% CO₂ in air) or hypoxic conditions (1% O₂, 5% CO₂ and 94% N₂) in a hypoxic chamber (ASTEC, Fukuoka, Japan). YC-1 (Sigma-Aldrich), NAC (Sigma-Aldrich) and Insulin (Wako, Osaka, Japan) were used at a final concentration of 10 µM, 5 mM and 500 ng/ml, respectively. High glucose medium was prepared using a 45% D-(+)-Glucose solution (Sigma-Aldrich) and the concentration was determined to be 10 g/L, 5 times higher than RPMI-1640 medium.