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. 2015 May 27;89(15):8026–8041. doi: 10.1128/JVI.00787-15

FIG 5.

FIG 5

Reduced autolysosome formation in DENV-infected cells. (A) Measurement of lysosomal activity in DENV-infected cells. Huh7 cells were mock infected or infected with DENV NGC (MOI = 5) for 36 h. In addition, mock-inoculated cells were subjected to nutrient deprivation, 100 nM BafA1, or full medium conditions for 6 h before harvesting. The cells were treated with Magic Red (20 μg/ml) for 2 h, fixed with 4% PFA, and further processed for flow cytometry-based image analysis. The data set represented here is an average of three independent experiments. The data were analyzed analogous to the description in the legend to Fig. 1B. (B) Colocalization of lysosomes with LC3-positive vesicles. Huh7 cells stably expressing the autophagic vesicle marker mCherry-LC3 were either mock treated or infected with DENV NGC (MOI = 5) for 36 h, respectively. The cells were fixed with 4% PFA and stained for lysosomes (Lamp2) and viral protein (NS4B). Samples were analyzed by confocal microscopy imaging. Images on the right represent magnifications of the regions highlighted in the corresponding merge images in the left. Note the colocalization of mCherry-LC3 punctae and lysosomes (marked with white arrow). Quantification of the total number of colocalizing spots in DENV-infected cells, as determined by signal overlap between mCherry-LC3 and the lysosomal marker Lamp2, was performed. For each condition, more than 50 cells were analyzed. The level of significance is measured by a Student t test and denoted as an asterisk (*, P ≤ 0.05). (C) Endolysosomal trafficking in DENV-infected cells. Huh7 cells were mock inoculated or infected with DENV NGC (MOI = 5) for 36 h. The cells were treated with DQ-BSA (20 μg/ml) for 12 h. Mock-inoculated cells were subjected to nutrient deprivation, 100 nM BafA1 treatment, or full medium conditions for 6 h before harvesting. The left panel shows examples of image-based flow cytometry detection of DQ-BSA; the right panel displays the relation of DQ-BSA vesicle areas and intensities. The data shown here represent the averages of three independent experiments.