Figure 1.
HCV affects proliferation, anchorage-independent growth, adhesion and migration of Huh7.5.1 cells.
(A) RT-PCR for the 5′UTR in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). (B) Protein expression levels of HCV core (upper panel), HCV NS3 (middle panel) and beta-actin (loading control, lower panel) in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). Immunoblots are representative of at least five independent experiments. (C) Proliferation rate was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD (bars) of five independent experiments. *P<0.001 versus Ctrl. (D) Adhesion test, and (E) cell migration assay. Data reported in the histograms (panels D and E) were expressed as the number of adherent or migrating cells × 103 compared with controls, considered as 100×103. Columns, means of three independent experiments; bars, ±S.D. P value was calculated versus the control cells: *P<0.001; **P<0.01; ***P<0.05. (F) Anchorage-independent growth was evaluated by colony formation assay in soft agar. After 28 days of incubation; colonies >0.15 mm in size were counted under microscope (×10 magnification). Data is expressed as means±SD (bars) of three independent experiments. *P<0.05 versus Ctrl.
Figure 2.
HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules.
(A) Immunofluorescence of paxillin (green), and alpha-actinin (red) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI (blue) was included to stain the nuclei. Magnification bar: 30 µ. (B) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. (C) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). (D) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. (E) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against beta-actin (as loading control). *P<0.001 versus Ctrl.
Figure 3.
HCV infection indirectly induces proliferation, invasion, activation and a pro-fibrogenic phenotype in LX-2 cells.
(A) Proliferation rate of LX-2 (NM) and LX-2 (CM) was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD (bars) of five independent experiments. *P<0.001 versus LX-2 (NM). (B) The invasive abilities of LX-2 (NM) and LX-2 (CM) were examined after 24 hrs by chamber assay. Spectrophotometrical analysis (optical density) was employed for quantification of the transmembrane cells. Histogram reports the mean value ±SD (bars) of three independent experiments. *P<0.01 versus LX-2 (NM). (C) Immunofluorescence of alpha-SMA (red) by confocal microscopy in LX-2 (NM) and LX-2 (CM) cells after 24 hrs stimulus. DAPI (blue) was included to stain the nuclei. Magnification bar: 30 µ. (D) Real-time PCR for the expression of alpha-SMA mRNA. The histogram reports the relative quantity of alpha-SMA mRNA normalized for actin. Values between +1 and –1 indicating invariant expression, are not shown. Data is statistically significant (*P≤0.05) for relative quantity greater than ±1.5. (E) Concentration of HA by ELISA assay in medium collected at time 0 and after 24 hrs of treatment from LX-2 (NM) and LX-2 (CM) cells. Histograms report the mean values (microg/L of HA) ±SD, (bars) of 4 independent experiments. *P<0.001; **P<0.01 versus LX-2 (NM).
Figure 4.
FAK siRNA counteracts HCV-dependent direct effects on Huh7.5.1 cells.
(A) Percentage of cell mortality and FAK silencing after 24 hrs from treatment of HCV-infected cells with different amounts of FAK siRNA with respect to the control siRNA considered as 0. Histograms represent % Trypan Blue positive cells and % of inhibition of FAK protein expression ± SD (bars). *P<0.001. (B) Protein expression levels of total FAK 24 hrs after siRNA transfection. Immunoblots are representative of at least four independent experiments. (C) Proliferation rate was evaluated as incorporation of BrdU performed after 24 hrs from siRNA transfection. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD (bars) of five independent experiments. *P<0.001. (D) Cell migration assay, and (E) adhesion test. Data reported in the histograms (panels c and d) is expressed as the number of adherent or migrating cells compared with controls, considered as 100×03. Columns, means of three independent experiments ±SD (bars). *P<0.001. (F) Anchorage-independent growth was evaluated by colony formation assay in soft agar. After 28 days of incubation, colonies >0.15 mm in size were counted under microscope (×10 magnification). Data reported in the histogram is expressed as means ±SD (bars) of three independent experiments. *P<0.01 versus Ctrl. (G) Immunofluorescence of paxillin (green), and alpha-actinin (red) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from siRNA transfection. DAPI (blue) was included to stain the nuclei. Magnification bar: 30 µ.
Figure 5.
HCV infection promotes physical interaction between FAK and phosphorylated paxillin and alpha-actinin.
(A) Immunoprecipitation analysis of FAK immunocomplexes in Ctrl and HCV Huh7.5.1 cells after 24 hrs from scramble or siFAK transfection. Western blotting of paxillin (upper panel) and alpha-actinin (middle panel) was shown. Actin was used as loading control of whole extracts (lower panel). Input proteins are reported in the last lane. All blots are representative of three independent experiments. (B) Immunoprecipitation analysis of tyrosine phosphorylation of paxillin (upper panel) and alpha-actinin (middle panel) in Ctrl and HCV Huh7.5.1 cells after 24 hrs from scramble or siFAK transfection. Actin was used as loading control of whole extracts (lower panel). Input proteins are reported in the last lane. All blots are representative of three independent experiments.
Figure 6.
FAK siRNA counteracts HCV-dependent indirect effects on LX-2 cells.
(A) Proliferation rate of LX-2 treated with scramble-NM, siFAK-NM, scramble-CM and siFAK-CM was evaluated as incorporation of BrdU performed at three different time points: 0 and 24 hrs. Quantitative data of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD (bars) of five independent experiments. *P<0.001 versus Ctrl. (B) The invasive abilities of LX-2 treated with scramble-NM, siFAK-NM, scramble-CM and siFAK-CM were examined after hrs by chamber assay. Spectrophotometrical analysis (optical density) was employed for quantification of the transmembrane cells. Data reported in the histogram is the mean value ±SD (bars) of three independent experiments. *P<0.01 versus scramble-NM; *P<0.05 versus scramble-CM. Immunofluorescence of alpha-SMA (red) by confocal microscopy in LX-2 treated with scramble-NM, siFAK-NM, scramble-CM and siFAK-CM after 24 hrs of conditioning. DAPI (blue) was included to stain the nuclei. Magnification bar: 30 µ. (C) Concentration of HA by ELISA assay in medium collected at time 0 and after 24 hrs of treatment of LX-2 with scramble-NM, siFAK-NM, scramble-CM and siFAK-CM. Histogram reports the mean values (microg/L of HA)±SD, (bars) of 4 independent experiments. *P<0.001 versus Ctrl. (D) Concentration of TNF-alpha by ELISA assay in medium collected at time 0 and after 24 hrs from transfection of Hhu7.5.1 cells with scramble and siFAK. Histograms report the mean values (microg/L of TNF-alpha) ±SD, (bars) of 4 independent experiments. *P<0.001. (E) Real-time PCR for the expression of TNF-alpha mRNA. The histogram reports the relative quantity of TNF-alpha mRNA normalized for actin. Values between +1 and –1 indicating invariant expression, are not shown. Data is statistically significant (*P≤0.05) for relative quantity greater than ±1.5.
Figure 7.
FAK siRNA enhance apoptotic response in HCV conditioned LX-2 cells.
(A) The histogram reports O.D. at 450 nm ±SD (bars) that provides a measure of cleaved PARP after 0, 6 and 24 hrs of LX-2 conditioning with NM and CM. *P<0.001. (B) The histogram reports O.D. at 450nm ±SD (bars) that provides a measure of PARP cleavage after 0 and 24 hrs of LX-2 conditioning with scramble-NM and siFAK-NM, scramble-CM and siFAK-CM. *P<0.001.