Figure 7: p140Cap negatively controls ERBB2-driven migratory ability and Rac GTPase activity.

(a,c,e,g) Representative images of Transwell migration assays. 10⁵ cells were left to migrate for 24 h in the presence or the absence of 15% FBS, fixed, stained and counted. Histograms represent on the y axes the fold increase (ratio between the number of cells migrated in the presence and in the absence of FBS), from three independent experiments, performed in triplicate. Error bar: s.e.m. (a) p140Cap over-expressing (oe p140) or mock SKBR3 (mock) cells. (c) SKBR3 cells transiently transfected with ON-TARGET plus human SRCIN1 small-interfering RNA (si p140) or ON-TARGET plus non-targeting siRNA (Dharmacon RNAi; si ctrl). This patented approach strongly prevents off-target effects. (e) MDA-MB-453 cells transiently transfected with ON-TARGET plus small-interfering RNA as in c. (g) Primary NeuT and p140 cancer cells. (b,d,f,h) Active Rac pull-down from cells like in (a,c,e,g). Eluted material (upper panels) and cell extracts (lower panels) run on 12% SDS–PAGE revealed with anti Rac antibodies. Histograms show the ratio between active and total Rac protein levels in arbitrary units (a.u.) from five independent experiments. Statistical significative differences were evaluated using umpaired t tests (*P<0.05; **P<0.01). Error bar: s.e.m. (i) Primary NeuT cells were grown in Matrigel/CollagenI 1:1 for 1 week, before seven days treatment with 80μM Rac1 inhibitor NSC23766 and acini immunostained for GM130 (green), beta1 integrin (red) and DAPI for nuclei. Scale bar, 50 μm. Histograms represent quantification of acina area (left) and polarity (right) from three independent experiments. Differences in acina area were evaluated using a Mann–Whitney non parametric t-test (***P<0.001). Error bar: s.e.m. (j) p140 primary cancer cells were infected with retroviral particles that express Rac1-V12 or empty vector (retro Ctrl). Cells were plated in Matrigel/Collagen I 1:1 and day 15 acinar structures were immunostained as in i. Scale bar, 50 μm. Quantification of acini area in a.u., percentage of polarized acina and percentage of acina with protrusions are reported. The values from two independent experiments are reported. Differences were evaluated using a Mann–Whitney non parametric t-test (***P<0.0001; *P<0.05). Error bar: s.e.m.