Journal: Nature Communications
Article Title: Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii
Figure Lengend Snippet: EV proteins and RNA are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded DNA (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different
Article Snippet: To degrade single-stranded DNA and RNA deprived of double-stranded regions, 0.4 μl of S1 nuclease (Thermo Fisher Scientific #EN0321) was added to 20 μl EVsR265 for 30 min at room temperature.
Techniques: Isolation, Infection, Activation Assay, Incubation