Figure 7. Adhesion strength is decreased in NEDD92/2 MEFs. A. Surface expression of a5b1 determined by FACs analysis in WT (grey) and NEDD9 2/2 (white) MEFs. B. a5b1 distribution in cells grown under 2D conditions. Arrows point to a5b1-positive focal adhesions. C. Fibronectin (FN) staining and Differential Interference Contrast (DIC) images of WT and 2/2 cells in 3D collagen gels. D. Fraction of detached fibronectin-coated beads versus pulling force. Adhesion strength was lower in NEDD92/2 MEFs (grey circles, n = 91) compared with WT MEFs (black circles, n = 103). E. The level of activated b1 (detected using b1 antibody clone 9EG7) normalized to a5b1 and expressed as fold change relative to the WT control. FACs data are the average 6 SEM from 5 experiments repeated on separate days. * p,0.05, *** p,0.001, Students’ t-test. doi:10.1371/journal.pone.0035058.g007 affinity for talin, thus reducing focal adhesion turnover [20]. Subsequent connections to the actin cytoskeleton allow the transmission of acto-myosin forces through the adhesion site which results in integrin activation. There was little evidence of activated b1 integrin co-localising with stress fibres in the NEDD92/2 MEFs. Thus a picture is emerging to suggest that molecules which regulate cell adhesion force to the ECM may determine the traction force required for cells to negotiate the surrounding 3D ECM. An important question this raises is whether NEDD9, similar to the function of vinculin [40], impacts the formation and contractility of actin stress fibres and/or, whether NEDD9 mechanically couples contractile forces to the focal adhesions. The role that adhesion proteins such as NEDD9 play in the regulation of cell migration in 3D contexts is still only poorly NEDD9 Stabilization of Focal Adhesions understood. Our results suggest that NEDD9 may stabilize focal adhesions and increase adhesion force to the extra-cellular matrix, providing new insight into the function of NEDD9 in 3D cell migration. NEDD9 expression is highly restricted [12] and the protein half-life is short and tightly regulated [36]. As migrating cells negotiate different environments in vivo the regulated expression of molecules such as NEDD9 may facilitate the necessary adhesive force required to traverse tissue barriers. Materials and Methods Cell culture and antibodies Mouse embryo fibroblasts were maintained in Dulbecco’s Modified Eagles Medium (DMEM) (Invitrogen) with 15% Foetal Bovine Serum (FBS) and supplemented with antibiotics (pen/ strep, Invitrogen). Immortalized mouse embryo fibroblasts (MEFS) were kindly supplied by Prof Erica Golemis (Fox Chase Cancer Center, Philadelphia USA). Briefly, wild-type and NEDD9 knockout mouse embryos were generated in the Golemis lab through cross-breeding of heterozygous and homozygous NEDD9 B57/BL6 mice [41]. Embryos were genotyped and wild- type and homozygous knockout MEFs were immortalized with the large T antigen of the SV40 virus. All experiments involving mice were preapproved by the Fox Chase Cancer Center Institutional Animal Care and Use Committee (protocol 06-1, PI Golemis). Growth of cells in 3D collagen gels, media for live imaging and invasion assays through 3D collagen gels have been previously described [42]. The following antibodies were used: anti-NEDD9 (ImmuQuest; clone 2G9 and Cell Signalling Technology; Clone 2G9), anti-FAK (Cell Signalling Technology), anti-phospho-FAK Tyr397 (Life Technologies), anti-Src (Millipore, clone GD11), anti- phosphor-Src Tyr418 (Life Technologies), anti-Myosin Light Chain 2 (Cell Signalling Technology), anti-phospho-Myosin Light Chain 2 Ser19 (Cell Signalling Technology), anti-p42/44 MAPK (Cell Signalling Technology), anti-phospho-p42/44 MAPK Thr202/Tyr204 (Cell Signalling Technology), rat anti-mouse b1 integrin mAb to detect activated b1 integrin (BD bioscience; clone 9EG7), rat anti-mouse a5b1 integrin mAb (Millipore; clone MAB2575), rat IgG isotype control (BD bioscience) and anti- fibronectin (Sigma-Aldrich); HRP-conjugated anti-mouse and anti-rabbit (Amersham and Biorad) and anti-rat Alexa-Fluor 647 conjugate (Molecular Probes, Invitrogen). Protein extraction, siRNA and immunoblotting Conditions of protein extraction from 2D cultures and immunoblotting were carried out as previously described [43]. To extract proteins from cells cultured in 3D collagen gels, gels were first treated with Type I Collagenase (Life Technologies) for 30 min at 37uC and centrifuged at 1000 rpm for 5 min at RT. The cell pellet was washed with 16 PBS then resuspended in lysis buffer and incubated on ice for 30 min. Lysates were centrifuged for 12 min at 13000 rpm at 4uC and the supernatant retained for analysis. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce Biotechnology IL, USA) and protein concentrations equalized prior to loading on gels. Control siRNA and custom-designed NEDD9 siRNA (targeting the sequence CCA GGA CAU UCG CAA CAA A) were both purchased from Dharmacon. 10 nM siRNA was transiently transfected twice at 24 h intervals, using Lipofectamine2000 (Life Technologies) as per manufacturer’s instructions. Live cell imaging, migration and rounding analysis Time-lapse images were captured using an ORCA ERG cooled CCD camera (Hamamatsu, SDR Clinical Technology NSW, PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35058