Fig. 8. Cell spreading on fibronectin is impaired in the absence of EHD1. Ehd1^+/+ MEF (A), Ehd1^–/– MEF (B), Ehd1^–/– MEF cells transfected with GFP-EHD1 (C) or Ehd1^–/– MEF cells transfected with GFP-EHD1 G65R (D) were plated on coverslips coated with 10 µg/ml fibronectin and allowed to spread for 3 hours in complete media. Cells were then fixed and stained with Cy3 wheat germ agglutinin (WGA; red) (A-D). Cell surface boundaries were outlined for 80-100 individual cells chosen randomly [Ehd1^+/+ MEF cells, Ehd1^–/– MEF cells and Ehd1^–/– cells transfected with GFP-EHD1 (green)], and 40 cells for Ehd1^–/– MEF cells transfected with GFP-EHD1 G65R (green). LSM 5 Pascal software was used to calculate the mean surface area of each population (see error bars for standard deviation) (E). One-tailed Student's t-tests were applied to test statistical significance of the data, with the P values for Ehd1^+/+ versus Ehd1^–/– MEF being P<0.001; Ehd1^+/+ versus Ehd1^–/– MEF transfected with GFP-EHD1 G65R being P<0.0001; Ehd1^–/– versus Ehd1^–/– MEF transfected with GFP-EHD1 being P<0.0001; and Ehd1^–/– MEF transfected with GFP-EHD1 versus Ehd1^–/– MEF transfected with GFP-EHD1 G65R being P<0.0001. This is a representative experiment from four independent experiments showing a similar trend. Bar, 10 µm.