Extended Data Figure 2. PlxnD1 mediates the endothelial cell response to fluid shear stress.

(a) Bovine aortic ECs (BAECs) were transfected with Scr or PlxnD1 siRNA and exposed to laminar fluid shear stress (12 dynes/cm²) using a parallel plate system for the indicated time periods. Phosphorylation of Akt (n=6), ERK1/2 (n=5) and eNOS (n=8) was determined by western blotting and quantified using Image Studio Lite Ver 5.2. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism.*p<0.05 relative to static condition; #p<0.05 relative to the respective siScr shear time point. (b) BAECs were transfected with Scr or PlxnD1 siRNA and exposed to atheroprotective shear stress for 24 hours. Cells were fixed and stained with phallodin and DAPI as well as antibodies to β-catenin to visualise actin stress fibres, nuclei and cell junctions, respectively. Quantification of alignment was performed using ImageJ; n>50cells over 4 biological replicates (for exact n, please refer to source data). The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism; ****p<0.0001