Fig 1. Analysis of homotypic fusion of phagophores.

(A) Electron microscopy of the HCV replicon cell. Open arrowheads and black arrows denote phagophore-like membrane structures and autophagosomes, respectively. The large black arrow denotes an autophagosome that appeared to be assembled via the fusion of several phagophores. N, nucleus. (B) Illustration of the in vitro homotypic fusion assay of phagophores. See text for details. (C) Analysis of homotypic fusion of phagophores in vitro. Huh7 cells were transfected with the expression plasmid for mEmerald-ATG5 (green) or mCherry-ATG5 (red). Two days after the DNA transfection, cells with or without nutrient starvation for one hour were lysed for the phagophore fusion assay in the presence (+ATP) or absence (-ATP) of ATP. Nutrient starvation was conducted by incubating cells in Hanks balanced salt solution (HBSS). HCV replicon cells were similarly transfected with the ATG5-expressing plasmids and harvested 2 days after transfection (Replicon) for the membrane fusion assay. For HCV infection studies, Huh7.5 cells were transfected with the ATG5-expressing plasmid and, one day after transfection, infected with HCV (m.o.i. = 1) for 1 day and then lysed for the in vitro phagophore fusion assay. The fusion results were analyzed by fluorescence microscopy. (D) Quantification of mEmerald-ATG5 and mCherry-ATG5 puncta shown in (C). (E) Quantification of the homotypic fusion efficiency of ATG5 puncta shown in (C).