70ix microscope

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.

Two different HCC cell lines were used in the study: Hep3B, with epithelial characteristics and moderate autocrine expression of TGF-β, and SNU449 cells, which show a mesenchymal phenotype and higher autocrine TGF-β expression [10 (link),11 (link)]. Both cell lines were obtained from the European Collection of Cell Cultures (ECACC) and were never used in the laboratory for longer than four months after receipt or resuscitation. 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 chronic TGF-β treatment, human recombinant TGF–β1 (Calbiochem, La Jolla, USA) was used at 2 ng/mL and replaced every 48 h. Stable transfection of the TGF-β Receptor I (TβRI) was performed as previously published [11 (link)]. Cells were observed under an Olympus 70iX microscope. Further information about targeted knock-down of TβRI and immunofluorescence analysis is included in Supplementary Materials and Methods .