Fig. 1.
Autophagy is induced in cells infected with IBV
(a) Lipidation of endogenous LC3 during IBV infection. H1299 cells were infected with IBV at MOI∼2 or incubated with UV-IBV. Protein lysates were harvested at the indicated time points and subjected to Western blot analysis using antibodies against LC3 and IBV N. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. The ratio of LC3-II to the corresponding β-actin band was determined and normalized to the IBV-infected 0 h sample. The experiment was repeated three times with similar results, and the result of one representative experiment is shown.
(b) H1299-GFP-LC3 cells were treated with 0.5 μM rapamycin or the same volume of DMSO for 5 h before harvested for Western blot with EGFP antibody. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. The ratio of GFP-LC3-II to the corresponding β-actin band was determined and normalized to the solvent control (−). The experiment was repeated three times with similar results, and the result of one representative experiment is shown.
(c) H1299-GFP-LC3 cells were infected with IBV as in (a) and subjected to Western blot using antibodies against EGFP and IBV N. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated. GFP-LC3II to actin ratio was determined as in (b) and normalized to the IBV-infected 0 h sample. The experiment was repeated three times with similar results, and the result of one representative experiment is shown.
(d) H1299-GFP-LC3 cells were treated as in (b) and fixed. Cell nuclei were stained with DAPI. Fluorescent images were captured with a confocal microscope. The square region was enlarged in the bottom panel to highlight the GFP-LC3II puncta. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. (e) H1299-GFP-LC3 cells were infected as in (c). Cells were harvested at the indicated time points, fixed and stained with antibody against IBV N (red). Nuclei were stained with DAPI. The experiment was repeated three times with similar results, and the result of one representative experiment is shown.