OSM increases clustering of P-selectin in clathrin-coated pits of human endothelial cells. (A) Density of P-selectin molecules on the surface of HUVECs 10 minutes after adding control buffer or the indicated agonist. (B) Internalization rate of P-selectin on HUVECs 10 minutes after adding buffer with or without or OSM. (C) Internalization rate of P-selectin on HUVECs 10 minutes after adding histamine-containing buffer with or without OSM. (D) Representative confocal immunofluorescence images of HUVECs 10 minutes after treatment with histamine, OSM, or both agonists. The sections were fixed, permeabilized, and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (blue) to visualize cell nuclei and with anti–P-selectin antibody (green) to visualize the distribution of P-selectin. Scale bar, 20 μm. (E) Quantification of the areas of P-selectin puncta. (F) Representative confocal immunofluorescence images of HUVECs 10 minutes after treatment with histamine or with histamine plus OSM. The sections were fixed, permeabilized, and incubated with anti–P-selectin antibody (green) and anti–α-adaptin antibody (red). Merged images revealed partial colocalization of α-adaptin with P-selectin (yellow). Scale bar, 20 μm. (G) Quantification of colocalization of P-selectin with α-adaptin. (H) Number of fixed human neutrophils, in isotonic or hypertonic medium with or without OSM, rolling on HUVECs at the indicated time. (I) Number of fixed human neutrophils, in isotonic or hypertonic medium with or without OSM, rolling on HUVECs at the indicated time after addition of histamine. (J) Number of fixed human neutrophils, in isotonic or hypertonic medium with or without OSM, rolling on immobilized purified P-selectin at the indicated time. The data in panels A-C,E,G represent the mean ± SEM from 3 to 8 experiments. The data in panels D and F are representative of 4 experiments. The data in panels H-J represent the mean ± SD from 3 experiments. *P < .05; **P < .01.