PI3K activity is required for stable adhesion and resistance to flow-induced detachment and deformation. (a and b) Accumulation assays (2 dynes/cm2 for 2 min) with U937 cells pretreated with vehicle or LY294002 (LY) or transfected with control or PI3K-γ siRNA. The extent of PI3K-γ knockdown by siRNA as determined by Western blotting was 85% (n = 3; see Fig. S5). The number of U937 cells that underwent arrest and remained adherent was quantified (n = 12 per group; three IEs; *, P < 0.001 relative to the control group). (b) Detachment assays with leukocytes (indicated) treated with PI3K inhibitors, 1 µM Wortmannin (Wort), 50 µM LY294002, and 100 µM AS605240 and transfected with control or PI3K-γ siRNA and CD8+ T cells isolated from wild-type and PI3K-γ knockout mice. The buffer contained Mn2+ (n = 10; three IEs; *, P < 0.05 relative to the control group). (c) Representative SEM images of 1 µM Wort-pretreated U937 cells and human monocytes adhered to VCAM-1 and then exposed to flow (2 dynes/cm2 for 2 min). Arrowheads indicate the extrusion of F-actin upstream of the cell body. (d) Wort perfusion induces cell displacement. U937 cells adhered to VCAM-1 were exposed to fluid flow (2 dynes/cm2 for 2 min). Vehicle or Wort (1 µM final concentration) was then added to the perfusion buffer (at time = 0 s). Representative sequential DIC images show Wort-induced cell displacement and distortion. Arrowheads indicate initial adhesion contact sites. (e) Displacement of U937 cells after exposure to fluid flow (2 dynes/cm2) for 40 s. Treatments are indicated (n = 3–10 cells per group; two IEs; *, P < 0.005; **, P < 0.0001). (f) Displacement over time of representative U937 cells. Error bars are means ± SD. Arrows indicate the direction of fluid flow.
