Figure 2.

Vesicle surface proteins are implicated in macrophage activation induced by EVs from ischaemic cardiomyocytes. (A) EV^CT or EV^ISCH were treated with 1 mg/mL trypsin for 10 minutes. Trypsin‐treated or untreated EVs were further incubated with macrophages for 24 hours. Nitrite production was determined and results are expressed as percentage of nitrite production over macrophages treated with EV^CT. ^§ P < 0.05 vs EV^ISCH (n = 4). (B) iNOS expression was evaluated in macrophages challenged with trypsin‐treated or untreated EV^CT or EV^ISCH for 24 hours, by WB. Graph depicts quantification levels. *P < 0.05 vs CT, ^# P < 0.05 vs EV^CT, ^§ P < 0.05 vs EV^ISCH (n = 5). (C) WGA‐stained macrophages (green) were incubated with PKH26‐labelled EV^CT or EV^ISCH (red) for 30 minutes, after which cells were fixed and visualized by fluorescence microscopy. Nuclei were stained with DAPI (blue). Scale bars 5 μm. (D) Macrophages were treated with 80 μmol/L dynasore for 30 minutes, followed by incubation with EV^CT or EV^ISCH for 3 hours. Medium was then replaced with EV‐depleted medium and cells were kept in culture for an additional 21 hours. Nitrite production was determined and results are expressed as percentage of nitrite production over macrophages treated with EV^CT for 24 hours. Cells challenged with EV^CT or EV^ISCH for 24 hours were used as control. ^### P < 0.001 vs EV^CT, ^§ P < 0.05 vs EV^ISCH (n = 5). (E) iNOS expression was evaluated in macrophages either treated or not with dynasore, followed by stimulation with EV^CT or EV^ISCH, by WB. Graph depicts quantification levels. *P < 0.05 vs CT, ^# P < 0.05 vs EV^CT, ^§ P < 0.05 vs EV^ISCH (n = 5)