NEDD9 Stabilization of Focal Adhesions Figure 8. Reduced stress fibres and activated b1 in NEDD92/2 MEFs in 3D. A. Activated b1 integrin (detected using b1 antibody clone 9EG7) and actin (detected with fluorescently-tagged phalloidin) immunostaining of cells grown in 2D conditions. Merged images show colour overlays of b1 (green) and actin (red). Arrows point to examples of active b1 integrin associated with the ends of actin stress fibres. Cells were imaged by wide-field microscopy. B. Activated b1 integrin and actin immunostaining of cells grown in 3D collagen gels. Boxed insets shows magnified regions containing actin stress fibres. Arrow heads point to actin stress fibres. Images represent single confocal z-slices. doi:10.1371/journal.pone.0035058.g008 Australia) and an Olympus IX81 inverted microscope equipped with an environmental chamber heated to 37uC. Transmitted light images were captured at indicated time intervals (406 objective). Cells undergoing division or apoptosis were excluded from analyses. Nuclear translocation was tracked in time-lapse image stacks using Metamorph V6.3 software (Molecular Devices). Random migration analyses were performed on sparsely plated cultures. To quantify cell migration behaviour, cell speeds and directional persistence were determined from the Mean Squared Displacement (MSD) of cells versus time as previously described [40]. Only elongated cells were tracked in the analysis of glioblastoma cell line 3D migration, to exclude apoptotic or mitotic cell populations. Cell morphologies in fixed 3D collagen gels were scored as either elongated (displaying extending protrusions and an elongated cell body) or rounded (displaying a rounded cell body and absence of protrusions). Cells were counterstained with propidium iodide and apoptotic cells excluded from morphological analyses. Immunofluorescence and FACs analysis Paxillin immunostaining of focal adhesions and focal adhesion morphometry analysis was performed as previously described [44]. Cells in 3D collagen gels were fixed, quenched, permeabilised and blocked overnight in 1% fatty-acid free BSA/1% donkey serum/ PBS. Gels were then incubated with primary antibodies in PBS/ 1% donkey serum, washed, followed by addition of fluorescently labelled secondary antibodies and TRITC-Phalloidin and after a final wash cells were imaged under (Olympus FV1000 Confocal Laser Scanning Microscope). FACs analysis of surface integrin PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35058