2.2. Cell culture

Human HCC-derived HuH7 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB0403) (Tokyo, Japan). HuH7 cells were maintained in RPMI-1640 medium (Sigma-Aldrich Co.) containing 10% fetal calf serum (FCS; Hyclone; GE Healthcare Life Sciences) at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. For Western blot analyses, the cells were seeded into 100-mm diameter dishes (6 × 10⁵ cells/dish) in RPMI-1640 medium containing 10% FCS. The medium was exchanged for serum-free RPMI-1640 medium after three days, and then the cells were used for experiments after 24 h. For the cell migration assay, the cells were seeded into 100-mm diameter dishes (4 × 10⁵ cells/dish) in RPMI-1640 medium containing 10% FCS for 4 days and then used for experiments.