Fig. 3.

SMAD4 is required for endothelial cell alignment, but not polarization, along the axis of laminar fluid flow. (A) Human coronary endothelial cells exposed to shear stress in a laminar flow chamber do not align, but still polarize their Golgi (green, GOLPH4⁺) against the direction of flow. VE-cadherin is shown in red; nuclei are labeled with DAPI (blue). (B) The absence of shear stress-induced alignment is quantified by plotting cell length versus width. Each dot is an individual cell. The green ovals indicate elongated cells. (C,D) Quantification of the angle between the nucleus, Golgi and flow showed a mild, but significant, decrease in polarization against flow. This is in contrast to the complete lack of alignment. Rose plots of measured angles are shown in C. n=140-300 cells per field of view. (E) Cell area is specifically increased under flow. (F) Quantification of proliferation rates through EdU incorporation. Smad4 depletion results in increased proliferation under flow (orbital). Data are mean±s.d. ns, non-significant; *P≤0.05; **P≤0.01; ***P≤0.001. EC, endothelial cell; FOV, field of view. Scale bars: 20 µm.