Supplemental Materials

This article contains the following supporting material:

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• Figure S1 - Characterization of endothelial cells that were transfected with cDNAs containing wt-IC1_GFP or IC1ΔCTD_GFP. The expression and localization of ICAM-1 or ICAM-1 mutant were imaged using EPI fluorescence microscopy. Bars, 10 μm.
• Figure S2 - Phospho-ERM is localized on the tip of growing microvilli in COS-7 cells expressing wt-IC1_GFP. COS-7 cells expressing wt-IC1_GFP were fixed and stained with anti-pERM antibodies followed by incubation with cy5-conjugated secondary antibody. The cells were then imaged using confocal microscopy equipped with 100X objective. Insets represent a magnified single image of the serial Z-sections. Bar, 10 μm.
• Figure S3 - Activation of HUVECs by TNF-α induces ICAM-1 and F-actin. (A) HUVECs were treated for the indicated times with TNF-α (10 ng/ml). The cells were then fixed and doubly stained with anti-ICAM-1 (R6.5) mAb followed by FITC-conjugated secondary antibody and TRITC-phalloidin. Cells were imaged using confocal microscopy with reconstitution in the z-axis. Bar, 10 μm. (B) The length of microvilli was determined using Olympus FV1000 Fluoview software (ver. 1.5). Values correspond to the arithmetic mean ąSD of a representative experiment of three independent experiments. *p<0.01, significantly different from TNF-α-untreated group (n = 3). (C) TNF-α-treated cells were fixed at the indicated time points, and the the cells were then stained with anti-ICAM-1 (R6.5) mAb followed by FITC-conjugated secondary antibody.
• Figure S4 - Dynamic movement of ICAM-1 clusters on the surface of COS-7 wt-IC1_GFP cells. COS-7 wt-IC1_GFP cells were incubated without (A) or with (B) CBR-LFA-1/2 mAb-activated PBL, and EPI fluorescence live cell time-lapse imaging was performed for 25 min at 37°C (also see Supplementary information, Movie 3 and 4). White arrowheads indicate time-dependent dynamic movement of GFP signals on the cell surface (A). Red arrowheads indicate rapid redistribution of GFP signals to form a ring-like cluster at the juncational interface of PBL-endothelial cells (B). Dotted red arrowheads indicate rapid joining of GFP signals at the ring-like clusters.