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Fig. 3. Inhibition of upstream protrusion is not due to increased RhoA activity and/or acto-myosin contractility. (A) Sparse PAE cells were treated for 30 minutes with 10 µM Y27632, a ROCK inhibitor, then exposed to flow for 5 minutes before being fixed and stained for actin. Notice the reduction in stress fibers, yet maintenance of polarized protrusion under flow (panel with arrow). (B) Kymograph demonstrating the effect of the ROCK/myosin light chain kinase inhibitor H7 (300 µM) on PAE cells in a wound, exposed to flow (arrow, 18 dyn/cm2). Notice that although the addition of H7 had a significant effect on downstream migration, it did not affect the flow-induced inhibition of upstream migration. (C,D) Sparse PAE cells expressing a dominant-negative form of RhoA (N19) (C) or C3-toxin (D) were exposed to flow for 5 minutes before being fixed and stained for actin. (E) At least 60 cells in each treatment were scored for the number and direction of protrusions. To visualize the degree of cell polarization the mean number of protrusions in the upstream or downstream directions (±45°) is presented. Error bars indicate s.e.m. Bar, 5 µm.
