The E1B19K protein plays an essential role in adenovirus-mediated autophagy.

<p>A. Representative electron micrographs of U-87 MG glioma cells infected with Delta-24-RGD or ΔE1B19K. Cells infected with Delta-24-RGD exhibited autophagolysosomal formation, as evidenced by double membrane organelles and cytoplasmic contain (arrows). B. Quantification of AVO after infection of U-87 MG cells with the indicated adenoviruses. Data are represented as mean ± SD of three independent experiments. *<i>P</i><0.01 versus Ad300-infected cells (two-tailed Student's t-test). C. Deletion of E1BK jeopardizes adenovirus-associated autophagy, as assessed by a significant decrease in the LC3 membrane localization. U87.GFP-LC3 cells were infected with Ad300 or ΔE1B19K (50 MOIs; 48 h). Shown are representative fluorescence images (Zeiss AxioImager 373), and mean ± SEM of three independent experiments of percentage of cells displaying GFP-LC3-positive puncta. *<i>P</i><0.001 versus Ad300-infected cells (two-tailed Student's t-test). D. Representative immunoblots from a panel of cancer cells after mock-infection or infection with the indicated adenoviruses show a decrease in the LC3-I to LC3-II conversion of cells infected with ΔE1B19K (indicated as LC3-II/LC3-I ratio). E. U87MG.GFP-LC3 cells were transfected with pcDNA.Flag, pcDNA.Flag-Beclin 1, or pcDNA.E1B19K, and 48 h later whole cell lysates were subjected to Western blot analyses to analyze the biochemical markers of autophagy: LC3-I to LC3-II conversion (indicated as LC3-II/LC3-I ratio) and p62 degradation. F. Representative fluorescence images of U-87 MG.GFP-LC3 after the indicated treatments are shown. Percentage of puncta-positive cells were quantified and data are represented as mean ± SEM from three independent experiments, from a minimum of 100 total cells per test. *<i>P</i><0.01 and ** <i>P</i><0.001 versus the mock group (two-tailed Student's t-test). G. HeLa cells were transfected with siBeclin or siControl and 24 h later, cells were mock- or Delta-24-RGD-infected (10 MOIs). After 48 h, cells were subjected to Western blot analysis to analyze LC3-I to LC3-II conversion (indicated as LC3-II/LC3-I ratio) and p62 degradation. E1B19K and Beclin 1 levels are shown, and tubulin levels were used as loading control.