Fig. 6

PABPC1/IFI27 regulate angiogenesis through exosome-transferred miR-21-5p regulation. A Representative images of exosomes by electron microscopy, NanoSight and western blotting (scale bars: 200 nm). B Transwell invasion and capillary tube formation assay of HUVECs treated with the indicated treatment and transfection (Scale bar: 50 µm). C HUVEC cells were incubated with PKH67-labeled exosomes from ESCCs for 24 h, and the green exosome signal was detected by confocal microscopy (scale bar, 20 µm).D MiRNA microarray was performed using PABPC1 or control transfected KYSE150 cells and cell-derived exosomes. E Expression of miR-21-5p was detected after the indicated transfection of KYSE150 and KYSE520 cells. F ESCCs transiently transfected with Cy3-tagged miR-21-5p were co-cultured with HUVEC cells for 48 h. Fluorescence microscopy was used to detect the red fluorescent signals in HUVECs (scale bar, 20 µm). G Expression of miR-21-5p was detected in ESCC-derived exosomes after the indicated transfection. H HUVECs were co-cultured with ESCCs concurrently transfected with Cy3-miR-21-5p and specific siRNAs targeting PABPC1 for 48 h. Fluorescence microscopy was used to detect fluorescent signals in HUVEC cells (scale bar, 10 µm). I Western blot analysis of PABPC1 expression in samples derived from miRNA pulldowns performed with nuclear, cytoplasmic, or exosomal ESCC lysates and the indicated biotinylated wildtype or mutated miR-21-5p; biotinylated poly(G) was used as a negative control. J. RIP assays with anti-PABPC1 antibody (or IgG as control) were performed on the cell or exosomal lysates from ESCCs. MiR-21-5p levels in immunoprecipitated samples were determined by qRT-PCR and were reported as percentages in respect to the input sample (% input). Data represent the mean ± SD of 3 separate determinations. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test