Fig. 4

SCAP-Exo improved the migration of HUVECs via Cdc42-mediated cytoskeletal reorganization. a F-actin immunofluorescence staining showed that actin cytoskeleton and filopodia formation (green) was obviously greater in SCAP-Exo-treated HUVECs than in control HUVECs. The number of filopodia per cell and the filopodia length of SCAP-Exo-treated HUVECs were higher than those of control HUVECs. Scale bar = 50 μm. b Western blot and pull-down assays showed that the expression levels of Cdc42-GTP and Cdc42 were elevated in the SCAP-Exo-treated HUVECs, while the expression levels of RhoA and Rac1 were not significantly changed. c Western blot and pull-down assays showed that siCdc42 significantly reduced Cdc42 and Cdc42-GTP expression in SCAP-Exo, while ML141 mainly decreased the expression level of Cdc42-GTP in SCAP-Exo. d The expression levels of Cdc42 and Cdc42-GTP were significantly upregulated in the SCAP^vehicle-Exo-treated HUVECs but not in the SCAP^siCdc42-Exo- and SCAP^ML141-Exo-treated HUVECs compared to the control HUVECs. e F-actin immunofluorescence staining showed that the actin cytoskeleton levels and the number and length of filopodia of HUVECs were higher in the SCAP^vehicle-Exo group than in the control group but were lower in the SCAP^siCdc42-Exo and SCAP^ML141-Exo groups than in the SCAP^vehicle-Exo group. Scale bar = 50 μm. f Representative images of the scratch wound healing assay showing that HUVEC migration in the SCAP^vehicle-Exo group was higher than that in the control group, while the migration ability of HUVECs in the SCAP^siCdc42-Exo and SCAP^ML141-Exo groups was lower than that in the SCAP^vehicle-Exo group at 24 h. Scale bar = 100 μm. g The in vitro tube formation assay showed that SCAP^siCdc42-Exo- and SCAP^ML141-Exo-treated HUVECs had a lower capacity to form vascular lumens than SCAP^vehicle-Exo-treated HUVECs. Scale bar = 100 μm. h H&E staining showed that there were fewer vascular lumens (yellow arrow) containing red blood cells in the SCAP^siCdc42-Exo and SCAP^ML141-Exo groups than in the SCAP^vehicle-Exo group. Scale bar = 50 μm. i Western blot and pull-down assays showed that SCAP-Exo had higher levels of Cdc42 expression but weaker Cdc42-GTP expression than SCAP. j Immunofluorescence staining showed that Cdc42-EGFP-labelled proteins derived from SCAP-Exo (green) were expressed steadily in the cytoplasm of HUVECs in primary culture as well as in P3 and P6 HUVECs. Scale bar = 100 μm. k Laser confocal microscopy image showing the co-localization of SCAP-Exo-derived Cdc42-mCherry (red) and Cdc42 (green) in the cytoplasm of HUVECs. The slides were counterstained with DAPI (blue). Scale bar = 50 μm. n = 5 in each group. **P < 0.01, ***P < 0.001. Error bars: mean ± SD