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tra12138-sup-0001-FigureS1.docWord document169KFigure S1: A representative interference reflection microscopy image of an NIH3T3 cell expressing wt DC-SIGN. DC-SIGN was labeled with primary 120507 mAb followed by secondary goat-anti-mouse AlexaFluor488 IgG. (left) IRM image; (middle) TIRFM image; (right) overlay of the IRM and TIRFM images. Bars = 10 µm. It gives a representative interference reflection microscopy (IRM) image of NIH3T3 cells expressing wt DC-SIGN. This figure is associated with the discussion of the lower limit of our counting method in Discussion. The IRM images give a measure of the distance between the ventral cell surface and the glass substrate so that the amount by which the evanescent intensity is decreased as one moves away from the substrate can be estimated and accounted for when comparing microdomain brightnesses to those for single molecules.
tra12138-sup-0002-FigureS2.docWord document167KFigure S2: Scatter plot of the sizes (FWHM) of DC-SIGN cell-surface domains with (green solid triangle) or without (red open triangle) DENV bound. It shows a scatter plot of DC-SIGN domains sizes, with or without DENV bound. This figure is associated with Results ‘Role of DC-SIGN microdomains in DENV infection’. We show that DC-SIGN micro/nanodomains are sufficient to capture viral particles, as the domains sizes after DENV bound are comparable with the sizes before DENV binding.
tra12138-sup-0003-FigureS3.docWord document497KFigure S3: DENV infection assay by FACS and confocal imaging. A) Percentages of cells infected after 24 h of incubation with DENV, measured by FACS. In the ‘DC-SIGN’ row, ‘−’ means cells not expressing DC-SIGN, i.e. wt NIH3T3 cells, and ‘+’ means NIH3T3 cells expressing DC-SIGN. In the ‘DENV’ row, ‘−’ means without incubation with DENV, ‘+’ means with incubation with DENV at MOI 1.57, and ‘++’ means incubation with DENV at MOI 15.7. B) wt NIH3T3 cells exhibited little infection after 72 h incubation with DENV at MOI 15. The yellow arrow points to a viral particle on the cell, while no productive infection was observed. Bar = 10 µm. C) NIH3T3 cells expressing DC-SIGN showed that almost every cell was productively infected after 24 h incubation with DENVs at MOI 15.7. Bar, 10 µm. D) 3D maximum intensity projection of confocal Z-stacks on DENV infected NIH3T3 cells expressing DC-SIGN. At 24 h after infection, massive viral protein replication was observed at the ER (left); at 48 h, viral particle assembly and transportation was observed in the cytoplasm (middle); at 72 h, many newly synthesized DENV particles were secreted into the extracellular space (right). For both FACS and confocal imaging, DENVs were stained with 2H2-AlexaFluor488 conjugates (against PrM protein), and DC-SIGN was stained with H-200 followed by anti-rabbit AlexaFluor647 (Fab')2. It shows representative DENV infection assay data for both FACS and confocal imaging. This figure is associated with Results ‘Role of DC-SIGN microdomains in DENV infection’. In Results we show that DC-SIGN microdomains capture DENVs within minutes of incubation, and this Figure further shows that DC-SIGN capturing of DENVs eventually leads to productive host cell infection.
tra12138-sup-0004-FigureS4.docWord document92KFigure S4: NIH3T3 cells with higher expression levels of DC-SIGN have more cell-associated DENV particles. Relative DC-SIGN expression levels were estimated from the mean fluorescence intensity of DC-SIGN staining on ventral cell surfaces by confocal imaging. The numbers of DENV particles was calculated by the Mosaic Particle Tracker 2D/3D plugin in ImageJ. It shows that cells with higher expression levels of DC-SIGN take in larger amount of viruses under identical incubation conditions. This figure is associated with Results ‘Role of DC-SIGN microdomains in DENV infection’. In Figure 6 we show that DENVs highly colocalized with cell-surface DC-SIGN microdomamins, and this supplemental figure shows that cells with higher expression levels of DC-SIGN take up more viral particles compared to cells with lower DC-SIGN expression levels, which further proves that DC-SIGN plays a major role in DENV uptake.

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