2.2. Cell Maintenance of Huh7 and Hep3B

Huh7 and Hep3B cells (HCC cell lines) were purchased from the Korean Cell Line Bank (Seoul, Korea). Huh7 cells were maintained in RPMI1640 with HEPES medium and Hep3B in DMEM/High glucose medium, both containing 10% fetal bovine serum. Monolayer cultures of Huh7 and Hep3B cells were incubated at 37 °C in an incubator with an atmosphere of 5% CO₂. When cell confluency reached 70% in 60 mm culture dishes or 96-well plates, the cells were incubated in serum-free medium for 24 h and then treated with various concentrations of brassinin for 48 h. AML-12 cells (mouse normal liver cell line) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to the manufacturer’s instructions.