Figure 5.

Quantification of hepatitis C virus (HCV) core and autophagy flux protein p62 expression in infected Huh-7.5 cells by flow cytometry. Infected cells were collected by trypsin-EDTA treatment and washed with phosphate-buffered saline (PBS). Infected cells were fixed for 10 minutes with 2% paraformaldehyde and permeabilized for 15 minutes using ice-cold methanol. For p62 detection, cells were incubated with a rabbit monoclonal antibody tagged with Alexa Fluor 488 (1:100) for 60 minutes. For detection of HCV core, cells were incubated with mouse monoclonal antibody (1:200) for 60 minutes. After three washes, cells were incubated with Alexa Fluor 488–conjugated secondary antibody (1:500) for 60 minutes. After these steps, cells were washed three times with PBS and analyzed separately by flow analysis. A: Percentage of core-positive and p62-positive cells were compared at each time points. B: Histogram of p62-positive cells from 0 to 30 days. P62 positivity decreases until day 12; after that p62 expression increases. C: Histogram showing the expression levels of HCV core. The number of core-positive cells gradually increases from 0 to 12 days. D: Expression levels of p62 and HCV NS3 in infected Huh-7.5 cultures by Western blot analysis. d, days; FITC-A, fluorescein isothiocyanate A.