Fig. 5. Effects of NO on lamellipodial VASP and cell shape in human embryonic fibroblasts (HEK cells). (a,b) Montage from time-lapse sequence of HEK cells transfected with WT GFP-VASP and S239A GFP-VASP respectively at the indicated times. NO donor was added at 25 minutes in panel a and 24 minutes in panel b. Last panel shows outline of cell from first panel. (c) Effects of NO on the localization of VASP in lamellipodia. The area of lamellipodia occupied by WT GFP-VASP was calculated just prior to addition of NO donor (control) and at the indicated times after NO addition, expressed as a percentage of the control value prior to NO addition. Values are the mean ± s.e.m.; n=7. The effect of NO was significant as determined by ANOVA, P<0.001. The inset shows an exponential decay curve fitted to the data; dotted lines are 95% confidence limits. The calculated half-life of reduction of VASP localization within lamellipodia after NO addition was 18.6 minutes. (d) As panel c, but from cells transfected with S239A GFP-VASP. No significant effect after NO donor addition was observed (ANOVA, P=0.32). Values are the mean ± s.e.m.; n=6. (e) Mean cell area normalized to that just prior to NO donor addition. Black bars, cells transfected with WT GFP-VASP, white bars cells transfected with S239A GFP-VASP. Areas were measured 30 minutes before (Control) and after (After NO donor) NO donor addition. Values are the mean ± s.e.m.; n=6. Decrease of area in cells transfected with WT GFP-VASP following NO donor addition was significantly reduced compared with control value (^***P<0.001, two-tailed t-test).