Figure 6

HCV proteins induced apoptosis in a caspase-dependent manner. A: Extent of apoptosis. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG. At 24 h p.i, uninfected and infected cells where fixed with an EtOH 70%-PBS solution, washed and stained with propidium iodide (PI) as explained in Material and Methods. The cell cycle was measure by flow cytometry. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: Activation of effector caspases. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of IPTG individually or in combination with a general caspase inhibitor, zVAD-fmk at 50 μM. Cell lysates from uninfected and infected cells were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes full-length (116 kDa) and cleavage-PARP (89 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). C: Caspase-8 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually or in combination with a caspase-8 inhibitor, zIEDT-fmk at 50 μM, in the presence (+) or absence (-) of IPTG. Uninfected and infected cell lysates were collected at 48 h p.i. and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes procaspase- (57 kDa) and active-caspase-8 (43 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). D: Caspase-9 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually in the presence (+) or absence (-) of IPTG or in combination with a caspase-9 inhibitor, zLEHD-fmk at 50 μM. Uninfected and infected cell lysates were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes the active-caspase 9 (37 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). Cells infected with the inducible VV-PKR were used as positive control.