Fig 4. Association, clustering and redistribution of membrane proteins.

HUVEC with or without treatment by TNF-α and CD9 siRNA were subjected to immunostaining for ICAM-1, tetraspanin CD9 and junctional adhesion molecule JAM-A, respectively. Images are taken by laser confocal microscopy (LSM) and projections in z are presented. (A) ICAM-1 is virtually absent from control cells, but is induced by TNF-α and forms a punctuate pattern of elongated or even kinked spots with a diameter of around 1 μm. They are distributed all over the cell surface with a preference for the perinuclear area. CD9, which is located at the cellular junctions in control cells becomes redistributed over the whole apical cell membrane upon cell activation—similar to the pattern of ICAM-1. JAM-A, which is often associated to CD9, exhibits the same behavior. In the activated cell state, there is a high degree of colocalization with both CD9 and JAM-A in the apical membrane clusters of ICAM-1—seen in the merged image as whitely spots. (B) Western blotting (WB) of CD9 in a siRNA-treated cell preparation as described in detail under methods section. Efficiency of siRNA-transfection was routinely controlled by WB for each umbilical vein preparation. (C) JAM-A immunoreactivity is hardly affected by CD9 siRNA-transfection; the expression level remains high, and the distribution still is mainly spread over the apical surface and not relocated to the cell junctions. CD9 protein level is vastly reduced as already confirmed in (B) by WB. The merged image demonstrates the reduced degree of clustering. Pictures shown are representative for more than 5 independent cell preparations.