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. 2013 Aug 1;126(15):3356–3369. doi: 10.1242/jcs.123547

Fig. 7.

Fig. 7.

p190RhoGAP spatially restricts pCofilinS3 and barbed ends amplitude for efficient protrusion formation. (A) Representative images of control- and p190RhoGAP-siRNA-treated MTLn3 cells stained for cofilin and pCofilinS3 showing cofilin and pCofilinS3 distribution at EGF-stimulated protrusions. Dotted lines in the enlarged images delineate the leading edge. Right panel: pCofilin and cofilin intensity profiles along the indicated line in the close up insert on the overlay image. (B) Quantification of cofilin and pCofilinS3 fluorescence intensity at the lamellipodium of control- and p190RhoGAP-siRNA-treated MTLn3 cells, stimulated with EGF. Values are normalized to those of control cells. Number of cells analyzed: control siRNA = 38 cells, p190RhoGAP siRNA = 35 cells. P-values obtained by comparison with the control siRNA value. (C) Representative images of barbed ends and F-actin staining in response to EGF stimulation in control and p190RhoGAP siRNA MTLn3 cells. (D) Radial sweep of white boxes in panel C. A white line is used to generate the quantification depicted in the right panel. Right panel: fluorescence intensity of barbed end measured from the cell edge (0 µm) into the cell (4 µm), through a region 5 µm wide, in control- and p190RhoGAP-siRNA-treated MTLn3 cells. Values are the means of 24 control cells (with three measurements per cell) and 21 (with three measurements per cell) p190RhoGAP siRNA cells. Black arrows point to the decrease in p190RhoGAP siRNA barbed end amplitude compared with that of the control. (E) Quantification of barbed end intensity in response to EGF at lamellipodia in control- and p190RhoGAP-siRNA-treated MTLn3 cells; n = 3 independent experiments with ∼35 cells per group. (F) Quantification of protrusive activity in response to EGF in control- and p190RhoGAP-siRNA-treated MTLn3 cells. Membrane protrusion is standardized to that at time 0. Number of cells analyzed: control siRNA = 26 cells, p190RhoGAP siRNA = 28 cells. Scale bars: 10 µm (main image); 1 µm (insert). Pseudocolor scale bar shows the barbed end intensity **P<0.01, ***P<0.001. Error bars indicate the s.e.m.