GFP-LC3 puncta formation assay with immunocytochemistry

GFP-LC3 lentivirus was used to generate Huh7 cells stably expressing GFP-LC3 by using Blasticidin S selection method [36]. GFP-LC3 Huh7 cells were inoculated with JC1 or lentiviruses expressing various viral proteins. Two days after infection, the cells were lysed for Western blotting or fixed for immunocytochemistry. The fixed cells were blocked by 1% BSA solution in PBS, and NS5A (Rabbit) or Flag (mouse) antibodies were treated to visualize the protein of interest. Cy5 labeled anti-mouse antibody was used as a secondary antibody, and Hoechst-33258 was used for nucleus staining. Microscopy images were obtained by Custom TCS SP5 II MP confocal microscope (Leica). The images were analyzed by Las AF program, and GFP-LC3 puncta were counted per each cell.