Fig. 4.

RGD-integrin binding domain of the P2Y₂ receptor modulates shear stress-mediated cell alignment and agonist-induced ASF formation. Cells were transduced with either P2Y₂ RGD WT (a, c–e) or P2Y₂ RGE mutant (b, g–j) receptors and subjected to shear stress for 6 h (a, b) or receptor agonists for 1 h (c–j). Epifluorescence images (×20 objective in a, b, and ×40 objective in c–j) show representative HUVEC monolayers that were fixed and stained with anti-HA antibody (Red), ActinGreen™488, and NucBlue^® for HA-tagged P2Y₂ receptor, actin, and the nucleus, respectively. Cells expressing the P2Y₂ RGD WT receptor show cell alignment (a) in the direction of flow (white arrows) compared to cells expressing the P2Y₂ RGE mutant receptors (b). Similarly, agonist-induced ASF formation is prominent in cells expressing P2Y₂ RGD WT receptors (d–f) compared to control (c) or cells expressing P2Y₂ RGE mutant receptors (g–j). Bar graphs (k–m) indicate quantification of ASF from multiple experiments; n = 7 experiments; *P ≤ 0.05; scale bars 10 μm