Fig. 5 | Nature Communications

Fig. 5

From: Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii

Fig. 5

EVs are rapidly and actively internalised by macrophages. a Macrophages were exposed to 10 μg of EVsR265, fixed with 4% PFA after 5, 15, 30, 60 and 120 min incubation and then immunostained using capsule-specific monoclonal antibody MAb 18B7. Images are maximum projections (21 z-stacks with 0.5 μm intervals). Note the change in signal from the cell periphery (arrowheads) to the cell body (arrows) in a time-dependent manner. Bar: 10 μm. b EV uptake increases in a time-dependent manner. Data are presented as scattered dot plots with lines representing their medians. Mean fluorescent intensities were normalised using the median value at 2 h as 100%. Data are representative of results from four independent experiments with a minimum of 25 macrophages analysed per sample per experiment. Unpaired Mann−Whitney tests where * (P ≤ 0.05), significant difference; ** (P ≤ 0.01), significant difference; and ns (P > 0.05), not significantly different. c Half time determination of EVs uptake by J774 macrophages. Data are presented as a sigmoidal non-linear regression curve (four-parameter logistic curve) with medians and errors (interquartile range) obtained from four independent experiments (from Fig. 5b). d The uptake of EVs by J774 macrophages is blocked by actin polymerisation inhibitors, cytochalasin D (MO+CytoD+EVsR265) and latrunculin A (MO+LatA+EVsR265) or a lipid raft-specific inhibitor methyl-β-cyclodextrin (MO+MbCD+EVsR265), which depletes cholesterol. Data are presented as scattered dot plots with lines representing their medians. Graph showing percentages of mean fluorescent intensities from macrophages alone (MO) or incubated with MAb 18B7-immunostained EVs isolated from R265 (MO+EVsR265) and were normalised to a median value obtained for MO+EVsR265. Data are representative of results from 2 to 5 independent experiments with a minimum of 25 macrophages analysed per sample per experiment. Unpaired Mann−Whitney tests where **** (P ≤ 0.0001), highly significant difference

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