Figure 1.
Expression of HCV core proteins in primary mouse hepatocytes reduce cell growth inhibition and apoptosis induced by TGF-β.
(A,B,C) Mouse hepatocytes obtained from livers of transgenic mice expressing or not HCV core proteins isolated from tumor (T) or cirrhotic (NT) tissues were treated with TGF-β for 48 h before determination of cell proliferation, estimated by BrDU incorporation (A), cell viability (B) or caspase 3 activity (C). (D) Cells were treated with TRAIL (20 ng/ml) for 18 h before determination of caspase3 activity. Results represent the mean+/−SD of triplicates from a representative experiment. * p≤0.05, ** p≤0.005, *** p≤0.0005.
Figure 2.
Expression of HCV core proteins in primary mouse hepatocytes increase EMT induced by TGF-β.
(A) Morphologic changes of mouse hepatocytes expressing or not HCV T core protein observed after 48 h of culture with or without TGF-β (2 ng/ml). (B) Hepatocytes isolated from transgenic mice expressing HCV core proteins were treated with TGF-β (2 ng/ml) or SB431542 (1 µM) for 48 h and expression of αSMA was examined by immunofluorescence using a αSMA antibody. Data are representative of three independent experiments. (C) Hepatocytes isolated from transgenic mice expressing HCV core proteins were treated with TGF-β or SB431542 for 48 h and expression of E-cadherin was determined by Western blotting. Anti-p38 western blotting was used as control loading. Data are representative of three independent experiments.
Figure 3.
TGF-β cytostatic responses in primary human hepatocytes expressing HCV core proteins.
Freshly isolated cells were infected with lentiviruses encoding the HCV core protein variants or an inverted core sequence as control (CTL) (A) Levels of core expression were estimated by Western blot analysis different time points after lentivirus transduction. (B, C) Determination of cell viability (B) or caspase 3 activity (C) was performed after 96 h of treatment with TGF-β (5 ng/ml). Results represent the mean+/−SD of triplicates from a representative experiment. * p≤0.05, *** p≤0.0005.
Figure 4.
TGF-β increases EMT in primary human hepatocytes expressing HCV core proteins.
(A) Expression of αSMA or Vimentin was estimated by immunofluorescence analysis after treatment with TGF-β (5 ng/ml) or SB431542 (1 µM). (B) Expression of Fibronectin or E-Cadherin was estimated by Western blot analysis in the same experimental conditions. Data are representative of three independent experiments.
Figure 5.
TGF-β responses in Huh7 cells stably expressing HCV core protein.
(A) Expression of HCV core protein determined by Western blot analysis. (B, C) Cells were treated with TGF-β (5 ng/ml) for 48 h before determination of cell viability (B) or caspase3 activity (C). Results represent the mean+/−SD of triplicates from a representative experiment. *** p≤0.0005 (D) Mitochondrial membrane potential (ΔΨm) was estimated by FACS analysis in cells treated with TGF-β for 48 h. After staining with DiOC6(3), cells with low fluorescence intensity corresponding to low (ΔΨm) were gated and their number expressed as a percentage of the total population. A representative experiment is shown. (E) Cells were treated with TGF-β for 48 h and E-cadherin or αSMA expression was assessed by immunofluorescence analysis. Data are representative of three independent experiments. (F) Comparative expression of HCV core proteins. Extracts from cultured cells expressing the core protein (Huh7, human or mouse primary hepatocytes) or from livers of HCV-related HCC patients were analyzed by Western blot. Anti-p38 western blotting was used as control loading.
Figure 6.
Smad activation is essential to induce TGF-β -mediated EMT.
(A) Huh7 cells were co-transfected GFP together with TbR1act or TbR1l45M act plasmids in the presence or absence of HCV core vector. Immunofluorescence analysis was performed 48 h later with an anti αSMA antibody. (B) Expression of TbR1act or TbR1l45M act and HCV core protein were assessed by Western blotting using anti-HA, anti-Flag or anti-core antibodies respectively.
Figure 7.
Smad3 depletion prevented TGF-β responses in Huh7 cells expressing or not the HCV core protein.
(A) Smad3 expression determined by Western blot analysis in four independent clones selected after stable transfection with pRetroSuper-shRNA-Smad3 plasmid. Anti-p38 antibody was used as control loading. (B) Different clones were transfected with the CAGA-luc reporter plasmid and treated or not with TGF-β (5 ng/ml) for 18 h before determination of luciferase activity. Results were normalized with renilla luciferase and represent the mean of triplicates+/−SD. (C, D) Different clones were treated with TGF-β for 48 h before determination of cell viability (C) or caspase3 activity (D). Results represent the mean+/−SD of triplicates from a representative experiment. * p≤0.05, *** p≤0.0005, NS : not significant. (E) Different clones were treated with TGF-β for 48 h and αSMA polymerization was estimated by immunofluorescence analysis.
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.