JCS02523 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Shear flow inhibits upstream protrusion in single cells. Sparse PAEC were fixed and stained for vinculin and actin, under no flow conditions or after 5 minutes of shear stress (arrow, 20 dyn/cm²).

• Supplemental Figure 2 - Fig. S2. Phosphorylation of p130CAS and FAK is non-polarized in the absence of flow. Double staining of PAEC for total (left column) and phosphorylated (middle column) p130CAS (pY165) and FAK (pY397). Ratio images (right column) indicate that the phosphorylation of these proteins is not polarized, in contrast with the polarized distribution induced by shear stress (for comparison, see Fig. 5). Bar, 5 mm.

• Movie 1 - Movie 1. Polarization of a single PAEC under flow. Phase-contrast time-lapse (1 minute between frames) of a single PAE cell. Flow (20 dyn/cm², from top) was applied after 30 frames. Notice the instantaneous arrest of upstream protrusion upon application of shear stress.

• Movie 2 - Movie 2. Cell migration into a wound is inhibited by flow even when contractility is inhibited. The first 21 frames are under static conditions. Flow (20 dyn/cm²) is from top to bottom. After 21 frames the contractility inhibitor H7 is added.

• Movie 3 - Movie 3. Constitutively active Rac-L61 is able to overcome flow-induced inhibition of upstream protrusion. Transfected cell expressing a constitutively active Rac1-L61 is marked with an arrow and asterisk on first frame. A white frame denotes beginning of flow (20 dyn/cm², from top).

• Movie 4 - Movie 4. Dominant-negative Dock180 inhibits lamellae protrusions in PAEC. PAE cells were transfected with GFP alone (WT) or with GFP plus DOCK180 (DOCK) or with GFP plus DOCK180-PS (DK-PS). The first frame shows the transfected cells and the movie in transmission light was taken at a frame every 20 seconds for 20 minutes and displayed at 10 frames per second. Bar, 10 mm.