Fig. 8
During EC adhesion and migration, CD93 is recycled through its intracellular domain. YFP-tagged CD93 or CD93∆C deletion mutant were transfected into HUVECs. a: Cells were detached from the plate, resuspended in complete growth medium, plated on the substrate, fixed at late phases of spreading, and analyzed by confocal microscopy. Exogenous proteins were imaged as yellow. Dotted lines indicate nucleus boundary. Scale bars are 7 μm. b: Quantitative analysis of CD93+ve vesicles per cell from ECs treated as in a. The vesicle numbers represent the mean ± SD of three independent experiments (n = 20 cells). ****P < 0.0001; unpaired t-test. c: Representative images of transfected HUVECs fixed at late phases of spreading. Cells were analyzed by confocal microscopy using anti-Rab5c antibodies. Exogenous CD93∆C was imaged as green. A dashed line indicates nucleus boundary. A wdc image between CD93∆C and Rab5c is shown. Scale bar, 15 μm. d: F-actin and wild type or mutant CD93 were imaged at 5 h after production of a double-sided scratch in the cell monolayer. Arrows indicate direction of migration and dotted lines indicate the migrating front. Overlay of stained cells is shown. Scale bar, 40 μm. High-magnification pictures of ECs in the migrating front within the squared area are shown as wdc images. In wdc images, scale bars are 15 μm. e: Quantification of CD93 and CD93∆C along the migrating front of transfected cells. Bars represent the percentage of fluorescence intensity of the control. Data are presented as the mean ± SD of three independent experiments (n = 7 transfected cells along the migrating edge). **P < 0.01; unpaired t-test