Fig. 5

Platelet-derived exosomes induce NET formation through modulating the Akt/mTOR-related autophagy pathway. PMNs were cocultured with exosomes isolated from the supernatant of platelets stimulated with PBS (PBS-Exo) or LPS (LPS-Exo) for 5 h. a ROS expression in PMNs was measured by flow cytometry with CM-H2DCFDA staining. b PMNs autophagy was assessed by staining with Cyto-ID autophagy tracer. c Immunofluorescence images showing LC3B expression (green) in PMNs. Nuclei were counterstained with Hoechst (blue). d, e PMNs were cocultured with PBS-Exo or LPS-Exo with or without wortmannin (150 nM), 3-MA (5 mM), bafilomycin A1 (1 μM), rapamycin (100 nM), and NAC (50 μg/mL). d The supernatant dsDNA concentration was quantified by PicoGreen. e NET formation was visualized by confocal microscopy with the following fluorescent dyes: Hoechst 33342 (blue) and anti-neutrophil elastase (anti-NE; red). f PMNs were cocultured with PBS-Exo or LPS-Exo in the presence or absence of MHY1485 (2 μM). The supernatant dsDNA concentration was quantified by PicoGreen. g Western blot of p-Akt, Akt, p-mTOR, and mTOR in PMNs after coculturing with exosomes. All results are representative of 3 independent experiments. Graphs represent means ± SEM; *P < 0.05, **P < 0.01 compared within two groups