Fig. 3.

HCC cell-secreted exosomal ANGPT2 is recycled by recipient HUVECs. a HUVECs were transfected with the pLV-EGFP-hRab5 plasmid for 48–72 h to express the Rab5-EGFP fusion protein and then cultured with ANGPT2-mCherry-expressing exosomes derived from HCC cells for 11 h. The confocal laser scanning revealed that ANGPT2-mCherry and Rab5-EGFP colocalized near the nucleus in HUVECs. Scale bar = 15 μm. b HUVECs were cultured with ANGPT2-mCherry-expressing exosomes derived from HCC cells for 12 h. The confocal laser scanning microscopy observed that ANGPT2-mCherry and Rab11-EGFP also colocalized in HUVECs by immunofluorescent staining using Rab11 antibody. Scale bar = 15 μm. c HUVECs were cultured with ANGPT2-mCherry-expressing exosomes derived from HCC cells for 12 h. The co-IP assay showed that exosomal ANGPT2-mCherry had interaction with Rab5 and Rab11 in HUVECs. d HUVECs were cultured with or without HCC cell-secreted exosomes for 6 h, then washed with PBS for 3 times and cultured with fresh medium supplemented with 10% exosome-depleted FBS for 12 h. Immunoblotting showed that ANGPT2-mCherry was positive in exosomes derived from HUVECs which had been cultured with ANGPT2-mCherry-expressing exosomes