Rap1b loss promotes transendothelial migration in 3D model. (A) Immunofluorescence images of ICAM-1 (red) and nuclei (blue) on surface of bEND.3 cells with or without stimulation with LPS (2 µg/ml) treatment for 18 h. Bar, 10 µm. Flow cytometry histogram showing ICAM expression on unstimulated and stimulated bEND.3 that were stained with anti—ICAM-1 and Alexaflour-488 conjugated secondary antibody (n = 3 independent experiments). (B) The bar graph represents number of cells per field adhered to bEND.3 grown on collagen and activated without or with LPS (mean ± SD, n = 4 independent experiments). (C) Representative images showing the neutrophil location after adhesion and locomotion onto LPS-activated bEND.3. Bar, 10 µm. Images show the location of neutrophils on the surface of junctions (arrows) and on bEND.3 cell body, a distance away from junctions (arrowheads). (D) The bar graph represents the percentage of cells present at bEND.3 junctions and the percentage of cells transmigrated underneath bEND.3. Mean ± SD; n = 4 independent experiments. **, P < 0.01; ***, P < 0.001; NS, not significant using unpaired Student’s t test.