Figure 3. Flow-induced PKN2 activation involves Piezo1, G_q/G₁₁, and PDK1.

(A–I) HUAECs were transfected with a control siRNA or siRNAs directed against PDK1 (A and G), PKN2 (C and D), Piezo1 (E), or P2Y₂ (H), or were left untransfected (B and F) or were pretreated or not with 100 nM YM-254890 (YM) (I) for 30 minutes. Thereafter, cells were exposed to laminar flow (15 dynes/cm²) for 15 minutes for the indicated durations (A, E, H, and I), or to 5 μM PS48 for 15 minutes for the indicated durations (B–D), or to 1 μM Yoda1 for 15 minutes for the indicated durations (F and G). Total and phosphorylated levels of the indicated proteins in lysates were determined by immunoblotting (A–C, E–I), and the NOx concentration was measured in the supernatant (D). Graphs in A, C, and G–I show densitometric analyses of the blots (n = 3 independent experiments). Data represent the mean ± SEM. **P ≤ 0.01 and ***P ≤ 0.001, by 1-way ANOVA with Tukey’s post hoc test.