MPO inhibition in microvesicles.

Erythrocyte-derived microvesicles from Jak2^V617F HC-EC mice were incubated for 1 hour with an irreversible MPO inhibitor (MPOi, PF06281355, resuspended in DMSO, Sigma-Aldrich) diluted in PBS (5 mol/L final concentration). Then, the same amount of annexin V–positive erythrocyte-derived microvesicles (JAK2^WT, JAK2^V617F, and JAK2^V617F with MPOi) were washed in PBS and centrifuged at 20,500 g for 2 hours. The pellet containing the microvesicles was then resuspended in endothelial cell basic medium (PromoCell). HUVECs (single donor; PromoCell, C-12200, lot 445Z011) were then incubated for 2 hours at 37°C with these microvesicles. At the end of the incubation, and without washing cells, ROS generation was assessed using CellROX, as described above. After rinsing with medium and paraformaldehyde (4%, 5 minutes), HUVECs were costained with DAPI (0.1 μg/mL, Sigma-Aldrich) in order to identify nuclei. Images were acquired using a Leica SP8 confocal microscope at ×400 magnification.