Rac1 localizes in ventral actin-rich rosettes and ARF6 is required for Rac1 activation and recruitment.

<p><b>(A)</b> MDA-MB-231 cells stably expressing ARF6T157N were plated on cross-linked gelatin, fixed and stained for F-actin and Rac1. Upper insets are higher magnifications of boxed regions. Lower insets show rosettes from another cell. Scale bars, 10 μm and 5 μm (insets). <b>(B)</b> ARF6 immunoblotting analysis of MDA-MB-231 cell lysates after indicated siRNA treatments. Anti-β-actin was used as loading control. <b>(C)</b> MDA-MB-231 cells were treated with the indicated siRNAs for 72 hours, plated on gelatin and then serum-starved for 12–16 hrs. After 15 min treatment with EGF, cells were fixed and stained for F-actin and Rac1. Insets are higher magnification of boxed regions. Scale bars, 10 μm. Arrowheads, peripheral F-actin-rich rosette. <b>(D)</b> Quantification of Rac1 fluorescence intensity profile was done along a 160-pixel line drawn perpendicularly to the cell edge as shown in insets. Intensity profiles along the line from at least 50 cells per condition from two independent experiments were averaged and normalized to the highest fluorescence intensity value in the siNT-treated cells. Comparison between the two mean peak values (corresponding to the cell edge) was made with a two-way ANOVA test. ***, P < 0.001 (compared to siNT-treated cells). <b>(E)</b> MDA-MB-231 cells were treated with the indicated siRNAs for 72 hrs, plated on gelatin, serum-starved and treated with EGF for 1 or 15 min. Levels of GTP-bound ARF6 (blue bars) and Rac1 (purple bars) in the different conditions were measured with G-LISA assay. Values are normalized mean ± SEM from replicate samples from two independent experiments. Comparisons were made with a Student’s t-test. **, P < 0.01, ***, P < 0.001 (compared with siNT-treated cells).