NEDD9 Stabilization of Focal Adhesions Figure 2. Paxillin phosphorylation is increased in NEDD9 2/2 MEFs. A. Paxillin (pax) and phospho-paxillin (ppax) immunostaining. Merged images show colour overlays of paxillin (green) and phospho-paxillin (red). Right hand panels show ratio images of paxillin phosphorylation. Red hues reflect regions of highest phosphorylated paxillin. Boxed insets shows magnified focal adhesions. B. Ratio of phosphorylated paxillin at focal adhesions in WT (n = 197 from 10 individual cells) and NEDD9 2/2 MEFs (n = 201 from 10 individual cells). ***p,0.0001, Students’ t-test. doi:10.1371/journal.pone.0035058.g002 collagen gels, we questioned whether NEDD9 may regulate the cell adhesion force to the extra-cellular matrix. We first examined the expression of integrin receptors and found little detectable expression of the a2 integrin sub-unit of collagen receptors (data not shown) as has been previously reported for MEFs [21]. By comparison, the cells were strongly positive for the a5b1 fibronectin receptor and indeed there was a striking increase in the expression of a5b1 on the NEDD9 2/2 MEFs (Figure 7A). Correspondingly, immunofluorescence analysis of the distribution of a5b1 in fixed cells revealed greater organization of a5b1 in focal adhesions (Figure 7B). Although the 3D cultures are collagen- based, fibronectin is present both in the added serum and can also Figure 3. NEDD9 2/2 MEFs have increased migration speed in 2D cell migration assays. A. MSD calculated from trajectories of WT (black squares), and NEDD92/2 (white squares) MEFs in 2D cell migration assays. Data are the average values from 3 independent experiments, n.45 cells tracked per experiment. B. Cell speed is significantly increased in NEDD92/2 MEFs (WT n = 150, 2/2 n = 143). C. MSD calculated from the trajectories of GFP-transfected NEDD92/2 MEFs (black squares) and GFP.NEDD9 transfected NEDD92/2 MEFs (white squares). D. GFP.NEDD9 expression significantly reduces the migration speed of NEDD92/2 MEFs. (n = 53 each). *** p,0.0001, Students’ t-test. doi:10.1371/journal.pone.0035058.g003 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35058