Hepg2

MCF7, HeLa, and A549 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). HepG2 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). MCF7 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum. HeLa and A549 cells were cultured in Dulbecco’s minimal essential medium (DMEM, Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum. HepG2 cells were cultured in Minimum Essential Medium supplemented with 10% fetal bovine serum. Culture conditions were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Cytotoxicity was tested on these four human cell lines (MCF7, HeLa, A549, and HepG2) using the EZ-CyTox cell viability assay kit (Dojindo Laboratories, Kumamoto, Japan). Briefly, 5 × 10³ cells /well were seeded in 96-well plates. After 24 h, cells were treated with compounds at the indicated concentrations. After 72 h of incubation, the EZ-CyTox cell viability assay kit was used. After 2 h of incubation, the absorbance was measured at 450 nm with a reference at 620 nm. Cell viability was calculated as a percentage of the control.

HepG2 (human hepatocellular carcinoma cell line) and AML12 (mouse hepatocyte cell line) were obtained from the American Type Culture Collection (ATCC). HepG2 and AML12 cells were cultured in Eagle’s minimum essential media and DMEM/F12 media, respectively, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37°C with 5% CO₂. HepG2 cells were transfected by Fndc5/Irisin and Tgfbr2 expression plasmids using Fugene 6 reagent according to the manufacturer’s instructions. FNDC5/Irisin and Tgfbr2 protein expressions were detected by western blotting analyses using 10% SDS page gels employing anti V5 tag antibody (Thermo Fisher) and anti Tgfbr2 monoclonal antibody (Santa Cruz Biotechnology), respectively. AML12 cells in 96 well plates (seeded 5000 cells per well) were treated with irisin (1 μg/ml or 2 μg/ml) at 24 hr. Seventy-two hr after transfection (HepG2) or irisin treatment (AML12), cell viability was determined using Cell Counting Kit-8 (Dojindo).