Liver Cancer-Derived Hepatitis C Virus Core Proteins Shift TGF-Beta Responses from Tumor Suppression to Epithelial-Mesenchymal Transition (original) (raw)
Figure 8
TGF-β responses in Huh7 cells expressing different levels of Smad3.
(A) Huh7-shRNA-Smad3 cells (Clone 3) were transfected with increasing amounts of Myc-Smad3 expression vector together with pCMV renilla luciferase. Smad3 protein expression was evaluated 24 h later by Western blot analysis using an anti-Myc antibody and loading was normalized with renilla luciferase expression. (B) Huh7-shRNA-Smad3 cells (Clone 3) were cotransfected with the CAGA-luc reporter plasmid and increasing amounts of Myc-Smad3 vector together with pCMV renilla luciferase. 24 h later, they were treated or not with different doses of TGF-β for 18 h before determination of luciferase activity. Results were normalized with renilla luciferase and represent the mean+/−SD of triplicates from a representative experiment. (C, D) Huh7-shRNA-Smad3 cells (Clone 3) were transfected with increasing amounts of Myc-Smad3 vector together with pGFP plasmid and sorted by FACS 24 h later on the basis of GFP expression. Cells were then cultured for 24 h and treated with different doses of TGF-β for 48 h before determination of cell viability (C) or caspase3 activity (D). * p≤0.05, ** p≤0.005, *** p≤0.0005, NS : not significant. (E) Huh7-shRNA-Smad3 cells (Clone 3) were transfected with increasing amounts of Myc-Smad3 vector together with pGFP plasmid and sorted 24 h later on the basis of GFP expression. αSMA expression was estimated by immunofluorescence analysis after treatment with TGF-β (1 ng/ml) for 48 h.