Fluid flow activates PI3K at the upstream cell membrane and in anchors. (a–d) U937 cells transfected with probes that bind to PIP₃ (AKT-PH-GFP) or PIP₂ (PLC-δ–PH-GFP). Cells were adhered to a VCAM-1–coated surface and imaged before and after exposure to fluid flow (2 dynes/cm² for 2 min). Images of representative cells at levels above the adhesion surface to approximately the midpoint, with the corresponding GFP fluorescence profile at the white dashed lines. A difference between upstream and downstream GFP intensity was only observed in cells transfected with AKT-PH-GFP after exposure to fluid flow (dashed red lines in c vs. d). (e) Quantification of GFP fluorescence (means ± SD; n = 9 for flow and 4 for static; three IEs; *, P < 0.001) MFI, mean fluorescence intensity. (f and g) Accumulation of the AKT-PH-GFP (PIP₃), but not PLC-δ–PH-GFP (PIP₂), probe in anchors. Images of collapsed z stacks (top and side views) of representative transfected U937 cells that were exposed to flow as in a–d. Each red dashed line is a reference line that was set at the downstream peak of AKT-PH or PLC-δ–PH-GFP probe fluorescence intensity. This line facilitates comparison to the peak fluorescence intensity at the upstream edge of the cell. Increased upstream fluorescence is seen only with the AKT-PH-GFP probe. The corresponding GFP fluorescence profiles within the yellow boxes are shown below. Solid arrowheads indicate anchors, and open arrowheads show regions of anchor insertion. Arrows indicate the direction of fluid flow. Bars, 10 µm.