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. 2024 Feb 7;15:1150. doi: 10.1038/s41467-024-45125-1

Fig. 1. EV fusion triggers inflammatory reactions.

Fig. 1

A Human blood (Healthy donors: n = 5) subjected to flow cytometry analysis post LPS-challenge are presented (left panel). The translocation efficiency of CD42b, a platelet activation marker, to blood cells from healthy and LPS-treated blood is depicted (right panel). Statistical analysis utilized ordinary one-way ANOVA with Tukey’s multiple comparisons test, results presented as box-and-whisker plots from minimum value and up to the maximum value, The error bars represent the standard error of the mean (SEM). B The schematic diagram illustrates the quantification of EV outer bound proteins and inner packed proteins, including their phosphorylation pattern (Created with BioRender.com). C THP-1 cells, stimulated with LPS (10 μg/ml) or PBS, underwent multi-cytokine membrane array analysis (upper panel). Mean pixel densities, calculated via image software, identified up-regulated cytokines (n = 1, measurement) denoted in a bar graph (lower panel). D LPS-stimulated THP-1 cells were subjected to multiplex immunoassay before and after EV lysis, revealing changes in selected inflammatory mediators. Statistical analysis employed ordinary one-way ANOVA unpaired t-test with Mann-Whitney test (The error bars represent the SEM; n = 9 of different measurements). E THP-1 cells, stimulated with LPS, were analyzed over time. Cell and EV lysates, separated on SDS-PAGE, were stained for phosphoproteins (left panel) and total protein content (right panel). The data represent a representative experiment from two independent experiments. F Lysates from LPS-stimulated THP-1 cells and LPS-EVs were separated on SDS-PAGE and then probed with anti-phospho-tyrosine antibody. The data represent a representative experiment from three independent experiments. G Immunoprecipitation of IL1R1 receptor from LPS-stimulated THP-1 cell lysates and LPS-EV lysates was performed. Samples were probed for pIRAK4 and MyD88 antibodies, indicating receptor-associated signaling. The data represent a representative experiment from three independent experiments. H Lysates from LPS-stimulated THP-1 cells and LPS-EVs were separated on SDS-PAGE and probed for pIRAK4, MyD88, pTRAF2, and TRADD antibodies, revealing in vitro signaling dynamics. The data represent a representative experiment from two independent experiments.