Mhcc97l

Human hepatocellular carcinoma cell line MHCC97L, PLC/PRF/5 and human normal hepatocyte line LO2 were subscribed from American Type Culture Collection (ATCC, Manassas, VA, USA) and preserved in liquid nitrogen. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with antibiotics (100 U/mL penicillin, 100 U/mL streptomycin) and 10% Fetal Bovine Serum (FBS), in a humidified incubator (Sanyo Electric, Osaka, Japan) at 37 °C containing 5% CO₂. Cells were collected by disposing with trypsin (Hyclone, USA) after the medium has been refreshed for twice. The cells in the logarithmic growth phase were used for dectcting the cell viability by methyl thiazolyl tetrazolium (MTT) assay. The MHCC97L and LO2 cells were treated with serial concentrations of wogonin for 24 h. At the end of the incubation, 10 μL of MTT solution(Sigma-Aldrich, St. Louis, Missouri, MO, USA) was added to each well and incubated for another 2.5 h. Absorbance was detected at wavelength of 570 nm after 100 μL of DMSO was used to dissolve formazan crystal.

HL7702, Hep3B, MHCC97L and MHCC97H cells were obtained from the Cobioer Bioscience Company (Nanjing, China), and HCCLM3 cells were obtained from China Center for Type Culture Collection (Wuhan, China). HL7702 cells were cultured in 1640 medium (Life Technology, Carlsbad, CA, USA), and Hep3B, MHCC97L, MHCC97H and HCCLM3 cells were cultured in high‐glucose DMEM (Life Technologies Corporation, Carlsbad, CA, USA) and supplemented with 10% FBS (Life Technologies Corporation) in a humidified atmosphere of 5% CO₂. For GSK3β inhibition, MHCC97L and HCCLM3 cells were firstly incubated with 1 or 4 μg/ml BS‐I (Sigma‐Aldrich, St Louis, MO, USA) for 6 hrs, and then, 0.2 μM CHIR99021 (Selleckchem, Houston, TX, USA) or 4 mM LiCl (Sigma‐Aldrich) was added to the medium.

Human HCC cell lines BEL-7402, SMMC-7721, PLC/PRF/5, MHCC-97L and immortalized normal liver cell line MIHA were used. BEL-7402, SMMC-7721 and MIHA were obtained from Shanghai Institute of Cell Biology (Shanghai, China). PLC/PRF/5 was purchased from American Type Culture Collection (Manassas, VA). MHCC-97L was a gift from Professor Z.Y. Tang at Fudan University (Shanghai, China). BEL-7402 and SMMC-7721 were maintained in Dulbecco's Modified Eagle Minimal Essential Medium (DMEM) with high glucose (GIBCO-BRL, Grand Island, NY). PLC/PRF/5 was maintained in MEM (GIBCO-BRL) with 1mM sodium pyruvate (Sigma Aldrich, St. Louis, MO), whereas MHCC-97L and MIHA were maintained in DMEM high-glucose with sodium pyruvate. All media were supplemented with 10% fetal bovine serum (FBS), penicillin at 100 units/mL and streptomycin at 100 μg/mL (Invitrogen, Gaithersburg, MO). All cell lines were cultured at 37°C in a humidified incubator with 5% carbon dioxide (CO₂). For normoxic conditions, the cells were incubated in normal incubator (considered as 20% O₂). For hypoxic experiments, the cells were cultured in hypoxic chamber supplemented with 1% O₂.