Immunofluorescence analysis of Sema4D-induced cytoskeletal rearrangement. In wild-type HUVECs (left panels), soluble Sema4D triggered dramatic cytoskeletal rearrangements, characteristic of an angiogenic endothelium. On Sema4D stimulation (20 minutes), we observed the disappearance of stress fibers, the formation of membrane ruffles (phalloidin-FITC [fluorescein isothiocyanate] staining, top row), the dismantling of focal adhesions, and the internalization of β-catenin (α-βcatenin staining, lower rows). Reduced expression of Plexin B1, obtained as a consequence of HUVEC transfection with siRNA specific for Plexin B1, resulted in a lack of response to Sema4D stimulation for the overall actin reorganization and for the β-catenin relocalization (right column). Images were captured with a 63×/1.32 oil objective lens.