Plc prf 5

Three different HCC cell lines, having different EMT phenotypes and TGF-β expression, were analyzed. Hep3B, PLC/PRF/5, and HLE cells were purchased by the European Collection of Cell Cultures (ECACC).
Hep3B and PLC/PRF/5 cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM), while HLE cells were grown in Roswell Park Memorial Institute (RPMI) medium. Both media were supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) l-glutamine, 1% (v/v) penicillin/streptomycin; all these products were provided by Sigma Aldrich (St. Louis, MI, USA).

PLC/PRF/5, Hep3B and SNU449 cells were obtained from the European Collection of Cell Cultures (ECACC). Huh7 and HLE cells were from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank). Cell lines were never used in the laboratory for longer than four months after receipt or resuscitation. Characteristics of all the cell lines are presented in Supplementary Table S5. All cells were maintained in DMEM media (Lonza, Basel, Switzerland) supplemented with 10% FBS (Sera Laboratories International Ltd, West Sussex, UK), Penicillin (100 U/ml), Streptomycin (100 μg/ml) and Amphotericin (2.5 μg/ml) and L-glutamine (2 mM). They were maintained in a humidified atmosphere at 37 °C, 5% CO₂. For chronical TGF-β treatment, human recombinant TGF–β1 (Calbiochem, La Jolla, USA) was used at 2 ng/ml in a medium and replaced every 48 hours. Cells were observed under an Olympus 70iX microscope. Methods for immunofluorescence, western blot analysis and flow cytometry analysis are included in Supplementary materials and methods.