Hepg2

HepG2 (human liver cancer cell line; ATCC, Manassas, VA, USA) cells and Huh7 (human hepatoma cell line, ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Welgene, Gyeongsangbuk-do, Korea) and RPMI 1640 medium (Welgene) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin-streptomycin). AML12 (mouse hepatocyte cell line, ATCC) cells were cultured in DMEM/F-12 medium (Welgene) containing an additional 40 ng/mL dexamethasone and 1% insulin-transferrin-selenium-pyruvate supplementation under the same conditions as other cells. These cells were grown in an incubator with 5% CO₂ at 37 °C. Transient transfection was performed using Superfect (QIAGEN, Hilden, Germany) and polyethylenimine (Polysciences, Inc., Warrington, PA, USA) transfection reagent in accordance with each manufacturer’s instructions. Cells were used for experiments at 80% confluence. An appropriate amount of empty vector was added to adjust total DNA amount used for each transfection to be 1 μg/well and co-transfected Nano as an internal control. Firefly luciferase activity was normalized to Nano-Glo (Promega, Madison, WI, USA) luciferase activity. ACEA and 2-AG were used to treat at a concentration of 10 μM for 12 h. GSK4716 was treated in HepG2 and AML12 cells at a concentration of 10 μM for 12 h. Luciferase activity was measured at 48 h after transfection.

HepG2 (a human hepatocellular carcinoma cell) cell lines were obtained from the Korea Cell Bank (Seoul, South Korea). HEK293 (a human embryonic kidney cell) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). HepG2 and HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Welgene, Gyeongsan-si, South Korea) supplemented with 10% fetal bovine serum (Welgene, Gyeongsan-si, South Korea) and 1% penicillin/streptomycin (Thermo, Rockford, IL, USA). Incubator gas tension was maintained at 1% O₂/5% CO₂ for hypoxic conditions and 21% O₂/5% CO₂ for normoxic conditions (VS-9000GC; Vision Scientific, Seoul, South Korea).

Human hepatocellular carcinoma(HCC) HepG2, Hep 3B,SK-Hep1 and normal hepatocytes Chang cells were obtained from American Type Culture Collection (ATCC). AMPK α WT and KO mouse embryonic fibroblast (MEF) cells were obtained from Dr. Joohuh Ha. HepG2, SK-Hep1, Chang and MEF cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic (Welgene, South Korea).