FIGURE LEGENDS
FIGURE 1. Anaphylactic patients EVs characterization. Seven different EV preparations from each experimental condition were analyzed by NTA. (A ) Transmission Electron Microscopy characterization of circulating EVs. Scale bar: 200 nm. Image depicts three representatives AnEVs. (B ) Representative NTA showed an average size particle of 200 nm, (C ) without significant differences between BEVs and AnEVs. (D ) The particle number per ml of AnEVs is slightly increased compared with BEVs. (E ) AnEVs were characterized by Western Blot. Panels show immunoblots from three representative patients. Bona fide EVs markers, such as CD63, TSG101, Syntenin-1 and CD9, were detected.
FIGURE 2. Proteomic profiling and differential analysis of AnEVs and BEVs . (A ) Venn diagram of brute data obtained by MS/MS representing the intersection between detected proteins in BEVs and AnEVs. (B ) Volcano plot showing all proteins identified. In blue color are showed these statistically significant (p>0.05) in the AnEVs (right side) and in the BEVs (left side). Access number (UniProt) for proteins of interest (CDC42, Ficolin-2, S100A9) are shown. Principal component analysis (C ) and unsupervised hierarchical clustering (D ) of plasma derived EVs.
FIGURE 3. CDC42, S100A9 and Ficolin-2 are increased in AnEVs.(A ) Upper panels represent the abundance protein levels expressed as the quantified ratio (AnEVs/BEVs and BEVs/BEVs) of a larger group of anaphylactic plasma paired samples. CDC42 (*p=0.0137, n=15, MW=21KDa), S100A9 (**p=0.0017, n=20, MW=14KDa), Ficolin-2 (**p=0.0092, n=26, MW=34KDa). (B ) Bottom panels show three representative immunoblots from patients for each protein of interest. MW: Molecular weight
FIGURE 4. Functional protein association networks in AnEVs.Classification of the main cellular localization (A ) and function (B ) of the protein panel based on the UniProt database. (C ) Top Canonical Pathways obtained by IPA and symbols of the molecules. (D ) Panel illustrate the network established between the dataset of proteins contained in the Ingenuity Canonical pathways. Each color indicates a different canonical pathway. Variations in thickness of the connective lines show the evidence of interactions among proteins.
FIGURE 5. Plasma derived EVs from anaphylactic patients induce loss of the endothelial monolayer resistance. HMVEC-Ls were incubated with 100µg/ml of purified EVs from anaphylactic patients. (A ) Images show a green PKH67 labeling of AnEVs after 5 hours of cell incubation (magnification 20X). (B ) Orthogonal image reveals PKH67-EVs around the nuclei. (C ) Graphic shows the change in TEER measurements after addition of BEVs and AnEVs (n=16 patients). 2 way ANOVA followed by the Bonferroni test was performed: **p= 0.0033, ****p≤ 0.0001 vs Vehicle (EBM + PBS); #p= 0.0285 vs BEVs). (D ) Individual representation of change in TEER measurements at 5 hours; Unpaired t test; #p= 0.0302 vs BEVs