Protein extraction and characterization of exosomes

Protein in HUVECs was extracted with RIPA buffer containing PMSF (Solarbio, Beijing, China). The supernatant of the lysis and exosome derived from HCT116 and SW480 was quantified with a BCA kit. Then, more details are provided below [11 (link)]. The following antibodies were used: anti-CD9 (Abcam, ab263019); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD34 (Proteintech, 14,486–1-AP); anti-Integrin β1 (Proteintech, 12,594–1-AP); anti-VEGFA (Proteintech,19,003–1-AP); anti-PDK2 (Abcepta, AP7039b); anti-Akt (Abcam, ab179463); anti-p-Akt (Ser473); anti-GAPDH (Proteintech, 60,004–1-Ig). IF and IHC was performed as previously described [11 (link)]. IF was performed using anti-CD34 (Proteintech, 14,486–1-AP). Then, the image of IF was obtained using the LSM880 confocal microscope system (Zeiss, Jena, Germany). IHC was performed using anti-CD31 (Proteintech, 11,265–1-AP) and anti-CD34 (Proteintech, 14,486–1-AP). The images of IHC were acquired using a fluorescence microscope system (Olympus, Tokyo, Japan).