ICAM-1 Clustering on Endothelial Cells Recruits VCAM-1
Figure 4
ICAM-1-induced VCAM-1 recruitment depends on lipid-rafts and actin polymerization and promotes ICAM-1-induced adhesion. (a) Anti-ICAM-1-antibody coated beads were allowed to adhere for 60 minutes to TNFα-stimulated HUVECs that were left untreated (CTRL) or were pretreated for 30 minutes with either cyclodextrin (CD; 3.8 M) or cytochalasin B (CytoB; 1 g/mL). Subsequently, cells were fixed and immunostained with ICAM-1-FITC and VCAM-1 Abs. VCAM-1 recruitment to ICAM-1 beads was quantified which showed that close to 80% of ICAM-1 beads which recruited ICAM-1 (set as 100%) also recruited VCAM-1. CD pretreatment reduced VCAM-1 recruitment with 40% and CytoB with 50%. Experiment was carried out three times. Data are mean ± SEM. . (b) Anti-ICAM-1-antibody coated beads were allowed to adhere for 60 minutes to HeLa cells that were transfected as indicated. Subsequently, cells were fixed and analyzed by confocal microscopy for number of ICAM-1 beads that adhered to one transfected cell. 50 transfected cells per experiment were analyzed and the experiment is carried out in duplicate. The experiment was carried out three times. Data are mean ± SEM. . (c) Anti-VCAM-1-antibody coated beads were allowed to adhere for 60 minutes to HeLa cells that were transfected as indicated. Subsequently, cells were fixed and analyzed by confocal microscopy for number of VCAM-1 beads that adhered to one transfected cell. 50 transfected cells per experiment were analyzed and the experiment is carried out in duplicate. The experiment was carried out three times. Data are mean ± SEM. . (d) Nondifferentiated HL60 or U937 cells were allowed to adhere for 60 minutes to HeLa cells that were transfected as indicated. Subsequently, cells were fixed and analyzed by confocal microscopy for number of VCAM-1 beads that adhered to one transfected cell. 50 transfected cells per experiment were analyzed and the experiment is carried out in duplicate. The experiment was carried out two times. Data are mean ± SEM. .
