AFM topographic and fluorescence imaging of PS-containing domains on stressed HUVECs. (A) HUVECs grown on glass substrates and treated with staurosporine as in Figure 2 (0.5 μM, 15 minutes) followed by topographic AFM imaging, as described in “Materials and methods.” The representative micrograph shows a cell undergoing retraction with filopodialike protrusions. (B) High-resolution scan of the boxed region in panel A. (C) Topographic profile of the retracted portion of the cell along the black line in panel B indicating that the heights of 3 individual filopodialike structures varied between ∼20 and 30 nm. (D) HUVECs were treated with staurosporine, then incubated live with 10 nM lactadherin-488 (green) and annexin A5-Cy3 (red) and imaged by confocal and differential interference contrast (grayscale) microscopy. A representative image shows that the cell body contained punctate structures that preferentially bound either lactadherin or annexin A5 (eg, green and red arrows), though some areas of apparently coincident (yellow) binding were also noted. Thin filopodialike structures were at the periphery and a subset strongly bound lactadherin, but not annexin A5 (see box in panel E). Some filopodia bound neither lactadherin nor annexin A5 (cyan arrow). (E) A higher magnification fluorescent image of the area indicated in panel D. (F) High resolution AFM topographical scanning image of the cells protrusions shown by fluorescence in panel E. (G) Topographic profile of the protrusions along the line indicated in panel F, with a mean diameter of ∼15 nm.