Fig. 3: Disturbed-flow-induced PKN1 activation and nuclear translocation is mediated by integrin α5β1.
a, HUAECs were plated on fibronectin (FN), collagen IV (Col.IV) or poly-l-lysine (PLL) and were kept under static conditions (−) or were exposed to disturbed flow (DF) for 60 min. Total and phosphorylated PKN1 was determined by immunoblotting (n = 3 independent experiments). b,c, HUAECs were exposed to DF for 30 min (b) or 3 h (c) or were kept under static conditions after pre-treatment for 30 min without or with the integrin α5β1 antagonist ATN-161 (10 μM). Total and phosphorylated PKN1 was determined by immunoblotting (b), and FOS/FOSB expression was analyzed by qRT–PCR (c) (n = 3 independent experiments). d, HUAECs on FN or PLL were exposed to static conditions or DF for 1 h, after which total and phosphorylated PKN1 distribution in the nucleus and cytoplasm was analyzed by immunoblotting (n = 3 independent experiments). e, HUAECs were exposed to DF for 1 h or kept under static conditions and then stained with antibodies against PKN1 (red) and p-PKN1 (green), along with DAPI (blue). The ratio of nuclear and cytoplasmic PKN1 or p-PKN1 was quantified (n = 3 independent experiments). Scale bar, 20 μm. f,g, Cross-sections of the inner (f,g) and outer (f) curvatures of aortic arches from wild-type (WT) (f,g) or EC-Itga5-KO (g) mice were stained with antibodies against phosphorylated PKN1 (green) and CD31 (purple) as well as with DAPI (red). The bar diagrams show percentage of phosphorylated PKN1-positive area per endothelial DAPI-positive area. Scale bar, 20 μm (n = 6 mice per group; at least three sections were analyzed per animal). h, Representative immunofluorescence confocal images of sections of the human aorta. Sections were stained with antibodies against phosphorylated PKN1 (purple), CD31 (green) and VCAM1 (red) as well as with DAPI (blue). Arrows indicate endothelial marker-positive cells. The Spearman correlation analysis showed a significant relationship (r = 0.7747, P = 0.0028) (n = 13 different patients; at least three sections per patient sample were analyzed). Scale bar, 20 µm. Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test (a–c), Mann–Whitney two-sided test (d,e) or unpaired two-sided t-test (f,g)). NS, not significant; Con., control; ATN., ATN-161.
