Fig. 6. EGF stimulation induces retraction in cells expressing an ArfGAP mutant of GIT1. A431 cells were stimulated for the indicated times with 100-200 ng/ml of EGF. (A) EGF-induced lamellipodia protrusion is inhibited in a cell expressing the ArfGAP defective p95-C2-GFP protein (asterisk), whereas large lamellipodia are formed (arrowheads) in two neighbouring non-transfected cells (white dots). Same fields are shown in the frames of the upper and lower rows, respectively. Bar, 10 µm. (B) Cells with contact-free edges expressing p95-C2-GFP (n=23), p95-GFP (n=20), p95-C-GFP (n=18), or GFP (n=18) were analyzed by time-lapse digital analysis, and scored during the first 15 minutes of stimulation with EGF for formation of ruffles. Bars represent the percentages of cells showing inhibition of EGF-induced ruffling. (C) Retraction of two cells expressing p95-C2-GFP (asterisks) during EGF stimulation. No strong retraction was evident in the non-transfected cell. Bar, 20 µm. (D) Retraction was scored 30 minutes after stimulation with EGF in cells with contact-free edges expressing p95-C2-GFP (n=24 with EGF; n=20 no EGF), p95-GFP (n=23), p95-C-GFP (n=18), or GFP (n=22). Bars represent the percentages of retracted cells. (E) A431 cells were treated with 10 µM Y-27632 for 45 minutes before stimulation with 200 ng/ml EGF for the indicated times. The asterisk indicates a transfected cell; arrows indicate retractions. No strong retraction (arrowheads) was evident in the non-transfected cells. Bar, 20 µm. (F) Cells with contact-free edges expressing p95-C2-GFP treated with Y-27632 (30 cells), with EGF (23 cells), or with Y-27632 followed by EGF (25 cells) were analyzed by time-lapse digital analysis. Retraction was scored 30 minutes after EGF (or mock) stimulation. Bars represent the percentage of retracted cells. In B,D,F the standard error of the percentages is shown. Statistical significance was assessed by the 
2 test, incorporating Yates' correction for continuity, with P<0.05 considered significant. In B and D the values obtained from cells transfected with each of the GIT1 constructs were compared with that from cells transfected with GFP alone. *P<0.001; **P<0.0005. In F there was no significant difference in terms of retraction after EGF stimulation between Y-27632 treated and untreated cells.