Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid
Fig 3
AdV induced membrane damage causes selective autophagy.
(A) Schematic representation of the experimental setup for quantitative immune fluorescence analysis. Cells grown on coverslips were infected with virus at 37°C followed by inoculum removal after 30min. Coverslips were collected and fixed at indicated time points for quantitative immunofluorescence analysis as detailed in SI. (B) U2OS cells were infected with WT fluorescently labeled viruses (red signal) and fixed at 30min post infection and stained with anti PVI antibodies (green signal) and anti Gal8 antibodies (cyan signal). The insets to the right show triple colocalization (AdV+VI+Gal8) indicated by arrows (white signal). (C) U2OS cells were infected with fluorescently labeled WT (black line), M1 (red line) or TS1 viruses (gray line) as outlined in (A). At each time points cells were fixed and stained for Gal8. The top panel shows the absolute number of Gal8 punctae per cell, the bottom panel the percentage of Gal8 positive particles. Error bars show cell-to-cell variations (n>10 cells; NS: no significant; *: P<0.05; **: P<0.01; ***: P<0.005 and errors bars are standard deviation). (D) Representative confocal images of cells infected with different AdV showing virus induced autophagy. Conditions are indicated to the left of each row. Cells were fixed at 30 minutes post infection and stained with anti-AdV (first column, red signal) and anti-LC3 (first and second columns, green signal). (E) Infected U2OS cells were analyzed by western blot at 30 min (T30) and 60 min (T60) post infection using LC3 and GAPDH specific antibodies. As a positive control cells were treated with 50μM of CQ during 3hours. The LC3 signal depicts unconjugated (LC3-I) or PE-conjugated (LC3-II) LC3. Note that upon PE conjugation LC3 has a higher mobility in SDS-PAGE. The ratio of LC3II/GAPDH normalized to the non-infected condition was determined and is given below the panel. (F) Experiment as in (C) stained with anti-LC3 depicting the absolute number of LC3 punctae per cell.