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. 2013 Dec;87(24):13397–13408. doi: 10.1128/JVI.02116-13

Fig 4.

Fig 4

EHDV2-IBA induces autophagy. (A to C) OK cells at 24 h postplating were either noninfected or infected with EHDV2-IBA. At indicated time points, cells were collected and processed for 12% SDS-PAGE (see Materials and Methods). Panel A depicts a typical immunoblot probed with anti-LC3, anti-p62, and antiactin (loading control) antibodies. In panel B the graph depicts the average ± SE of the fold changes in the LC3-II/LC3-I ratios under the indicated conditions relative to the LC3-II/LC3-I ratio of uninfected cells at 12 hpi (taken as 1; n = 3). *, P < 0.05. The graph in panel C depicts the average ± SE of the fold changes in the p62/actin ratio under the indicated conditions relative to the p62/actin ratio of uninfected cells at 12 hpi (taken as 1; n = 3). **, P < 0.01. (D) A stable cell line of OK cells expressing LC3-GFP (generated as described in Materials and Methods) was infected with EHDV2-IBA (MOI of 1; 48 h). Infected and noninfected cells were lysed, and lysates were separated by 10% SDS-PAGE. The top panel is a representative immunoblot probed with anti-GFP and antiactin antibodies. For the experiments shown in the middle panels, LC3-GFP-expressing cells were plated on glass-bottomed 35-mm dishes, and at 24 h after plating cells were either left uninfected or infected with EHDV2-IBA. At 72 h after infection, cells were fixed, permeabilized, stained with anti-NS3 and Alexa Fluor-555 goat anti-mouse antibodies, and imaged by spinning-disk confocal microscopy. Panels depict a 3D rendition of confocal z-stacks (0.3 μm between steps) of infected (lower panel) or noninfected (upper panel) cells. Grid, 10 μm. For the experiments show in the bottom panels, LC3-GFP-expressing OK cells were cultured and infected as described above. Beginning at 36 hpi, cells were imaged by spinning-disk confocal time-lapse microscopy. Staining of cells cultured and infected in this manner with anti-NS3 antibodies yielded at least 90% of infected cells (data not shown). Scale bar, 10 μm. (E) OK cells cultured and plated as described for panel A were either untreated or incubated with UV-irradiated (UV-irr) EHDV2-IBA (for the indicated time periods). The panel shows a representative immunoblot of lysates of such cells, separated by 12% SDS-PAGE and probed with antibodies against LC3, p62, NS3, and actin.