Figure 2

Phenotypic characterization of extracellular vesicles (EVs). (A) Cell viability of VSMCs supplemented or not with FBS was assessed by annexin V/propidium iodide double staining. n = 9 per group. (B) EV concentrations are expressed as means for each size for the VSMC^PCSK9-EVs (dashed line) and VSMC^WT-EVs (solid line). EV concentrations were calculated as marginal means from negative binomial regression models. n = 6 derived from 6 different experiments. (C) p-value and False Discovery Rate p-value of the comparisons of each size EV for the entire 30–700 nm size range are reported. n = 6 derived from 6 different experiments. (D) Representative ultrastructural images of EVs by TEM. For both, the main image shows an EV whose size is compatible with intermediate EVs, and the insert shows an EV whose size is compatible with small EVs (arrow). Scale bar = 100 nm. (E) The expression of tetraspanins CD9 and CD63 was evaluated by flow cytometry analysis in EVs. Representative panels of three independent experiments. (F) Protein expression of β1-integrin and Alix as assessed by WB in EVs. Representative blots of three independent experiments. (G) PCSK9 expression in VSMC^WT-EVs and VSMC^PCSK9-EVs through WB analysis. Representative blot of three independent experiments. VSMC^PCSK9-EVs are those released by VSMC^PCSK9 and VSMC^WT-EVs are those released by VSMCs^WT. EV, extracellular vesicles; FBS, foetal bovine serum; VSMC^PCSK9-EVs, EVs released by VSMCs overexpressing PCSK9; VSMC^WT-EVs, EVs released by VSMCs wildtype; TEM, Transmission electron microscopy; WB, Western blot.