Fig 1.
The system providing laminar flow or disturbed flow.
(A) The whole system for providing different flows. (B) The structure in flow chamber to provide laminar flow contained a slip of glass, a silicone membrane with rectangular space and a coverslip. (C) The structure of vertical step to provide disturbed flow is consist of a slip of glass, two silicone membranes with different sizes of rectangular space and a coverslip.
Fig 2.
The diagrammatic sketch of image analysis.
The original fluorescence image of BAECs (A) were added Pseudo color (B), then subtracted the background(C) and enhanced the edge of cell (D). After that, the image was divided into 50×50 parts (E) to calculate and eliminate the interference of nucleus (F).
Fig 3.
Polarized Rac activation in BAECs was caused by laminar flow but not disturbed flow.
The image of BAECs transfected with PAK-PBD-GFP biosensor to represent the Rac activation (Left panel) upon laminar flow (A, n = 12) or disturbed flow (B, n = 5) application were dealt by our imaging analysis software (Right panel). The 3D spatiotemporal map (C) of averaged single cell images showed an obvious Rac activation at the downstream of BAECs from laminar flow group (Above), but not disturbed flow group (Below). After that, the data of the thirtieth minute from laminar flow (Above) and disturbed flow (Below) were fitted by logistic curve (D). The arrow indicated the main flow direction.
Table 1.
The parameters from fitting curve in different groups.
Fig 4.
Shear stress caused a polarized Rac activation in BAECs after pre-treatment of ML-7 and CytoD but eliminated by NOCO.
The images of BAECs transfected with biosensor (left panel) pretreated with ML-7(A, n = 7), CytoD(B, n = 6) or NOCO(C, n = 8) upon laminar flow application were dealt by the imaging analysis(right panel). The 3D spatiotemporal map (D) of averaged single cell images showed an obvious shear stress-induced Rac activation at the downstream of BAECs from ML-7 (Above) and CytoD group (Middle), but not NOCO (Below). After that, the data of the thirtieth minute from three groups were fitted by logistic curve. Above was the ML-7 group, middle was CytoD and below was the NOCO group (E). The arrow indicated the main flow direction.
Fig 5.
Shear stress caused a polarized Rac activation after pre-treatment of BA but eliminated by CHO.
The images of transfected BAECs (left panel) pretreated with BA (A, n = 5) or CHO (B, n = 5) upon laminar flow application were dealt by the imaging analysis (right panel). The 3D spatiotemporal map (C) of averaged single cell images showed an obvious shear stress-induced Rac activation at the downstream of BAECs from BA (Above) group, but not CHO group (Below). After that, the data of the thirtieth minute from BA group (Above) and CHO group(Below) is extracted and fitted by logistic curve(D). The arrow indicated the main flow direction.
Fig 6.
The model of Rac activation under laminar flow and disturbed flow.
(A) The static condition before shear stress application. (B)Under laminar flow, cell membrane deformed along the flow and transmitted force to the downstream edges of cells with the help of cytoskeleton, especially microtubule, and then active Rac.(C)While under disturbed flow, the cell membrane and cytoskeletal fibers changed randomly without any obvious spatial direction.