Figure 3.

Exosomal EPHA2 promotes endothelial cell angiogenesis via kinase-dependent forward signaling pathway. A Uptake of HM-Exos by endothelial cells can enhance EPHA2 phosphorylation at Tyr588, whereas HM-Exos from EPHA2-silenced cells failed to induce an upregulation of EPHA2 Tyr588 phosphorylation (Left). EPHA2-rich exosomes from HEK-293T cells can induce a dramatic increase in EPHA2 Tyr588 phosphorylation in endothelial cells (Middle). HM breast cancer cells pre-treated with ALW-II-41-27, an inhibitor of EPHA2, can inhibit EPHA2 phosphorylation at Tyr588 when the cells were treated with HM-Exos (Right). B ALW-II-41-27 treated HM-Exos could not promote the tube formation of endothelial cells. C Schematic diagram of the structure of EPHA2 and its mutants. Full-length EPHA2 and its three mutants EPHA2-ΔL, EPHA2-D739N and EPHA2-S897A were cloned into pCDH-mCherry vector (Left). The expression of EPHA2 and its mutants in cell lysates and exosomes was detected by Western blotting. β-actin was used as a loading control (Right). D, E Exosomes carrying EPHA2 and EPHA2-S897A, but not EPHA2-ΔL and EPHA2-D739N, promote the tube-forming ability of endothelial cells and the sprouting capacity of arterial rings. Data are expressed as mean ± SD, and all experiments were repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001 and ns P > 0.05 indicate no statistical significance. Scale bar: 200 μm.