Analysis of the progression of phagophores into autophagosomes in HCV replicon cells.

<p>(A) Colocalization analysis of ATG5 (red), LC3 (green) and ER (blue) in HCV replicon cells. HCV replicon cells stably expressing GFP-LC3 were transfected with the mCherry-ATG5-expressing plasmid for one day and then with the control siRNA or siSTX7 for two days. Cells were then stained with ER Tracker Blue. (B) Live cell imaging analysis. HCV replicon cells (upper panels) and nutrient-starved Huh7 cells (lower panels) that stably expressed LC3-GFP were transfected with the mCherry-ATG5-expressing plasmid for 2 days and then stained with ER Tracker Blue for 40 minutes before imaging. The HCV replicon cells were observed for 40 mins with images taken once every 8 minutes. Huh7 cells were starved for 1 hour and observed for 10 minutes with images taken once every 2 minutes. Noe that, in HCV replicon cells, the two mCheery-ATG5 puncta denoted by arrows were associated with the ER and became entirely GFP-LC3-positive (i.e., yellow in color) at the 16-minute time point. The GFP-LC3 puncta did not separate from the mCherry-ATG5 puncta until at the 40-minute time point. In contrast, all three mCherry-ATG5 puncta in nutrient-starved Huh7 cells became GFP-LC3-positive at the 2-4-minute time points and were separated from GFP-LC3 puncta at the 8-10-minute time points. (C) Quantification of the average time length required for LC3 puncta to separate from ATG5 puncta. More than 10 puncta were observed for each cell type.