Formation of the autophagy elongation complex in Huh7 cells.

A. Detection of the ATG5-12 conjugate by Western blot in mock (UI) and JFH1 infected Huh7 cells at more than 90% using an anti-ATG5 antibody. HCV infection and autophagosome accumulation were detected using anti-NS3 and anti-LC3 antibodies, respectively. β-actin represents loading control. B. In situ ATG5-12/16L1 complex formation was analyzed using PLA in JFH1-infected at more than 90% or uninfected cells. Cells were labeled for ATG5-12 and ATG16L1 using anti-ATG5 and anti-ATG16L1 respectively. CTL represents negative control lacking anti-ATG5 antibody. Nuclei were counterstained with DAPI (blue). Scale bar, 20μm. C. The frequency of PLA signals were significantly higher in both JFH1-infected and uninfected cells compared to control cells (CTL) (based on the count in 40 cells for each condition) (P<0.0001, 1way ANOVA).