Fig. 3

Notch receptor activation by oscillatory fluid flow in human cerebral microvascular endothelial cells (hCMEC/d3). A mRNA level expression of Notch receptors upon exposure of hCMEC/d3 to disturbed flow (n = 3). Notch3 and Notch4 become prominent as endothelial cells are exposed to oscillatory flow for 24 h, while Notch1 and Notch2 expressions were not significantly regulated by altered fluid flow. mRNA fold values in parallel and oscillatory flow were calculated relative to the static control. All data were normalized with GAPDH expression and are given as relative to static control. B hCMEC/d3 exposed to oscillatory flow at 15 dyn/cm² for 24 h resulted in the overexpression of NICD3, which was localized to the nucleus. NICD4 was also overexpressed in cells exposed to oscillatory flow, but cytoplasmic localization was more prominent. NICD1 was very faintly expressed in cells exposed to oscillatory flow, but was detected in mean fluorescence intensity (MFI) analysis. DAPI (blue) was used to counterstain nuclei (scale bar 20 µM, magnification ×40). C MFI was plotted as the average fluorescence intensity ± SD of five microscopic fields per flow condition and from three biological replicates. D, E DAPT and RO4929097 efficiently prevented NICD3 expression in endothelial cells in the presence of continuing altered flow. F Representative western blot of NICD3 protein present in proteins isolated from cells exposed to static (lane 3), parallel (lane 2), and oscillatory (lane 1) shear stress conditions. GAPDH was considered as the loading control. PF indicates parallel uniform shear stress and OF represents oscillatory shear stress. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus respective static or parallel uniform shear-treated groups. ns not significant