Investigating NF-κB Signaling in HCC Cell Lines

HCC cell lines, Huh-7(Japanese Cancer Research Bank) and HepG2 (American Type Culture Collection) were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicilin, and 100 µg/ml streptomycin(Sigma-Aldrich) at 37°C in a 5% CO₂ humidified incubator. Human soluble RANKL was purchased from PeproTech (NJ, USA). Helenalin, a specific NF-κB inhibitor, was obtained from Santa Cruz Biotechnology (CA, USA). For experiments, cells were incubated with 100 ng/ml RANKL or the appropriate negative control (phosphate buffer solution; PBS) for different periods of time before which some were pretreated with1 µM Helenalin or PBS for 60 minutes according to previously published works [22] (link), [26] (link).