Inhibition of PI3K blocks the accumulation of PAK-PBD and F-actin in anchors and their insertion sites. (a and b) U937 cells transfected with PAK-PBD-YFP were pretreated with 1 µM Wort (30 min), adhered under static conditions to VCAM-1 (2 min), and then exposed to fluid flow (2 dynes/cm2 for 2 min). Confocal z stacks were obtained just before and after exposure to flow. (a) Optical slices at the level of anchor insertion (open arrowheads) showed that Wort inhibited PAK-PBD-YFP accumulation. Solid arrowheads indicate anchors. (b) Quantification of upstream versus downstream PAK-PBD-YFP accumulation (means ± SD; n = 5 cells per group; three IEs). MFI, mean fluorescence intensity. (c) Lifeact-GFP–transfected U937 cells were adhered to VCAM-1 and exposed to fluid flow (2 dynes/cm2). Wort was then added to the perfusion buffer (at time = 0 s). Sequential images show a gradual diminution of F-actin in upstream anchor insertions (open arrowheads). Closed arrowheads indicate anchors. See Video 9. Arrows indicate the direction of fluid flow. Bars, 10 µm.
