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