FIG 3 .

IFN-β treatment reduces the number of double-membrane structures formed by EAV nsp2-3, while protein levels and nsp2-3 cleavage efficiency are not affected. (A) Schematic overview of the experimental setup. (B) HuH-7/tetR/HA-nsp2-3GFP cells were analyzed by flow cytometry for GFP fluorescence after 24 h of the indicated treatments. (C) Levels of the indicated proteins in HuH-7/tetR/HA-nsp2-3GFP cells were analyzed 24 h after the indicated treatments, using Western blotting. The expected position of the HA-nsp2-3GFP precursor is indicated. (D) Example of a mosaic map (right) of a single mesh of an EM grid (tetracycline-treated HuH-7/tetR/HA-nsp2-3GFP cells) composed of 1,164 images (2,048 by 2,048 pixels each) acquired at 6,800× magnification and binning 2, which corresponded to a pixel size of 3.2 nm. The closeup (left) was extracted from the mosaic map as indicated. Coloring represents annotations of nuclei (blue ovals) and EAV nsp2-3-induced DMS (green ovals) in this mesh. Scale bars represent 500 nm (left) and 20 µm (right), respectively. (E) In four independent experiments, the number of cell profiles positive for DMS (DMS⁺) was quantified as well as the number of cell profiles containing a nucleus (Nuclei). Multiple mosaic maps were analyzed for each sample. Ratios are calculated as the number of DMS⁺ cell profiles divided by the number of cell profiles containing a nucleus, and P values were calculated using chi-square tests for each experiment. *, P < 0.05; **, P < 0.01. n.s., not significant. The average reduction over 4 experiments was 27% ± 7% (P = 0.001).