Fig. 4
From: Conformational alteration in glycan induces phospholipase Cβ1 activation and angiogenesis
The angiogenesis enhanced by GHCer in EVs competed with PLCβ1 for TRAX binding. A Immunohistochemical staining of tissue sections with breast cancer using VK9 mAb. Red arrows indicate vessel formation. B Morphology and dimension of the EVs secreted from MCF-7 cells. The EVs were placed on carbon-coated copper grids, stained with uranyl acetate, and examined by transmission electron microscopy. Scale bar: 50 nm. C Immunogold staining of the isolated EVs using VK9 or anti-SSEA3Cer antibodies, followed by gold particle-conjugated secondary antibody (2nd Ab). Scale bar: 50 nm. D Matrigel (500 μL) mixed with EVs (30 μg), EVs (30 μg) + VK9 (5 μg), GHCer (16.2 μg), GHCer (16.2 μg) + VK9 (5 μg), or SSEA3Cer (14.7 μg) was injected subcutaneously to the flank of mice (n = 5). On day 14, Matrigel plugs were dissected and photographed, and the hemoglobin concentration was determined. **p < 0.01, ***p < 0.001, and ****p < 0.0001. E In vivo neovascularization of Matrigel plug induced by GHCer in EVs was examined by H&E staining and quantified for blood vessel density. *p < 0.05 and ***p < 0.001. F FRET assay for the colocalization of TRAX and PLCβ1 in HUVECs after incubation with EVs. HUVEC were incubated with PBS, EVs (100 μg), EVs + VK9 (5 μg), or EVs + isotype antibody for 16 h. The result shown here as mean ± SD is one representative from three independent experiments