Smad3 depletion prevented TGF-β responses in Huh7 cells expressing or not the HCV core protein.

<p>(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.</p

Expression of HCV core proteins in primary mouse hepatocytes increase EMT induced by TGF-β.

<p>(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.</p

TGF-β increases EMT in primary human hepatocytes expressing HCV core proteins.

<p>(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.</p

Expression of HCV core proteins in primary mouse hepatocytes reduce cell growth inhibition and apoptosis induced by TGF-β.

<p>(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.</p

Smad activation is essential to induce TGF-β -mediated EMT.

<p>(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.</p

TGF-β responses in Huh7 cells expressing different levels of Smad3.

<p>(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.</p

Principal mixed cryoglobulinemia (MC) manifestations present in the 75 HCV patients with MC (MC-HCV).

<p>Principal mixed cryoglobulinemia (MC) manifestations present in the 75 HCV patients with MC (MC-HCV).</p

Identification of a reference miRNA (internal control) for relative quantification of miRNA of interest: expression levels of Let-7d (panel A) and miR-16 (panel B) in HCV patients and controls.

<p>Identification of a reference miRNA (internal control) for relative quantification of miRNA of interest: expression levels of Let-7d (panel A) and miR-16 (panel B) in HCV patients and controls.</p

Main clinical and laboratory data of 35 healthy subjects (HS) and 167 HCV-chronically infected patients with mixed cryoglobulinemia (MC-HCV) or HCV-related non-Hodgkin’s lymphoma (NHL-HCV) or without MC or NHL (HCV).

<p>Results are presented as mean± standard deviation;</p><p>ns, not significant;</p>∧<p>ALT, alanine aminotransferase;</p><p>ULN, upper limit of normal.</p>#<p>Complement C3, normal values: 83 to 177 mg/dL;</p>‡<p>Complement C4, normal values: 20 to 150 mg/dL;</p>†<p>Rheumatoid Factor, normal values: <25 IU/mL.</p>*<p>HS vs MC-HCV; HS vs NHL-HCV; HCV vs MC-HCV.</p>**<p>HS vs MC-HCV; HS vs NHL-HCV; HCV vs MC-HCV; HCV vs NHL-HCV.</p>***<p>HCV vs MC-HCV.</p><p>°HS or HCV vs MC-HCV or NHL-HCV.</p

Expression levels of miR-26b in HS, HBV, HCV, MC-HCV and NHL-HCV groups and in a subgroup of MC-HCV patients after therapy-induced clearance of the viral infection and complete clinical response (MC post Tp).

<p>Expression levels of miR-26b in HS, HBV, HCV, MC-HCV and NHL-HCV groups and in a subgroup of MC-HCV patients after therapy-induced clearance of the viral infection and complete clinical response (MC post Tp).</p