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. 2015 Jul 22;89(19):9962–9973. doi: 10.1128/JVI.00577-15

FIG 1.

FIG 1

Establishment of the HCV cell-to-cell transmission assay. (A) Confocal analysis of the anti-HCV core immunofluorescence (red) in HCVcc JFH-1-infected Huh7.5-GFP-MAVS cells at 48, 72, and 96 h p.i. Nuclei are in blue. Bars, 25 μm. (B) Titration assay of the AR3a blocking antibody. Huh7.5-GFP-MAVS cells were infected with HCVcc and incubated with increased amounts of AR3a for 72 h p.i. The graph represents the mean + standard error of the mean (SEM) percentage of HCVcc infectivity relative to the no-antibody condition and countable FFU obtained from two independent experiments. (C) Number of HCVcc-infected Huh7.5-GFP-MAVS cells per focus in the presence of increased amounts of AR3a at 72 h p.i. In the scatter plot, each dot represents number of HCVcc-infected cells per focus, and the results correspond to the median of two experiments performed in duplicate. Ten images for each experimental condition were taken and counted. Horizontal lines represent the median of all foci studied. Significances are determined by the Kruskal-Wallis test followed by Dunn's test. (D) Confocal analysis of HCVcc-infected Huh7.5-GFP-MAVS cells incubated with complete DMEM, 1% agarose overlay, or 50 μg/ml of AR3a in complete DMEM at 72 and 96 h p.i. The perimeters of the foci are displayed using a red line. Nuclei are in blue. Bars, 75 μm. (E) Time course of the HCVcc infection under different culture conditions. Huh7.5-GFP-MAVS cells were infected with HCVcc and maintained in culture with complete DMEM (black), 50 μg/ml AR3A (red), or 1% agarose (blue) for up to 96 h p.i. The graph shows the mean + SEM number of foci per well at 48, 72, and 96 h p.i. from one experiment performed in triplicate. At 96 h p.i., the number of foci under the DMEM condition was not determined (nd) because of the generalized infection observed in all triplicates. (F) Focus size comparison between 1% agarose and 50 μg/ml AR3a at 72 and 96 h p.i. The graph depicts the mean + SEM from 3 experiments made in duplicate. Significance is given by the Mann-Whitney test.