Sema3E treatment causes dynamic changes in the actin cytoskeleton and cell collapse, which is inhibited by PlexinD1 or SH3BP1 knockdown. (A) Live cell imaging in control, PlexinD1, and SH3BP1 siRNA-transfected HUVECs after Sema3E treatment. Representative DIC images were taken from time-lapse videos of cells at different time points after Sema3E treatment. Control cells underwent obvious morphological changes and exhibited cell collapse in response to Sema3E treatment, whereas PlexinD1- and SH3BP1-depleted cells did not display any changes (see Videos 1, 2, and 3 for the entire video sequence). (B) Quantification of live imaging. Cell size at different time points was measure and normalized to time point 0. **, P < 0.001. Error bars indicate SEM. (C) Actin staining in control, PlexinD1, or SH3BP1 siRNA–transfected HUVECs treated with Sema3E at different time points. Cells transfected with the corresponding siRNA were treated with Sema3E, fixed, and stained with phalloidin (green). In untreated cells, well-organized actin networks with lamellipodia and F-actin stress fibers were seen. After 10 and 20 min of exposure to Sema3E, control cells lost their shape and saw a disruption of F-actin stress fibers. Sema3E treatment of PlexinD1 and SH3BP1 siRNA–transfected cells exhibited an organized network with lamellipodia (yellow arrows) and F-actin stress fibers (white arrows) at every time point. Blue, DAPI. Bar, 10 µm. (D) Quantification of changes in the presence of lamellipodia and stress actin fibers upon Sema3E treatment. In control cells, the number of cells with lamellipodia and stress fibers was significantly reduced after 10 and 20 min of Sema3E treatment. PlexinD1- and SH3BP1-transfected cells did not show significant changes in the presence of lamellipodia and actin stress fibers at 10 and 20 min from ligand introduction. n = 3. *, P < 0.01; **, P < 0.001. Error bars indicate SEM.