dialyzed FBS; a switch in the media reduces cellular proliferation and thymidine incorporation over 90% while maintaining cell viability [33,34]. An in vitro wound healing assay was performed as previously described [35]. In brief, near confluent cultures of rabbit adult fibroblasts were scraped making an ,1-cm wide denuded area, then either stimulated with cytokines {EGF (1 nM) or PDGF (200 nM)} or with control vehicle only; photographs were taken at 0 h and 48 h and the relative distance traveled by the cells at the acellular front was determined by computer-assisted image analysis. Markings on the plates ensured measurement at the same site of the photographs at each time point. The distance migrated was then expressed as a percentage of the control distance migrated within each experiment, which also facilitated inter-experiment comparisons. Measuring Traction of Individual Rabbit Adult Skin- derived Fibroblasts Traction, the cellular force that an individual adherent cell exerts on its substrate, was determined using traction force microscopy [36]. In this technique, scattered fibroblasts are plated on a collagen matrix into which are embedded fluorescently-labelled microbeads. Fibroblast cells attach to the matrix and, in keeping with their physiology, begin to apply a traction force to it, resulting in micro-displacements of the embedded beads. The quantity of displacement is measurable and can be analyzed through computer-assisted algorithms. Fibroblast populations treated with multiple agents can be compared by analyzing a sufficient number of cells from each experimental condition. Briefly, a polyacrylamide gel disk (120 mm thick, 10 mm in diameter) embedded with 0.2 mm green fluorescent micro-beads (Molecular Probes, Eugene, OR) was made and attached to the bottom of a 35 mm glass dish (with a 14 mm circular inner glass area) (MatTek, Ashland, MA) that was consecutively pretreated with 0.1M sodium hydroxide, 3-aminopropyltrimethoxysilane, and 0.5% glutaraldehyde. The gel surface was then pretreated with Sulfo-Sanpah (Pierce, Rockford, IL) and coated with 200 ml of 100 mg/ml collagen type I. Rabbit adult and fetal fibroblasts in passage 2–4 were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution as mentioned above. Initial experiments measured the traction force exerted by adult fibroblasts relative to that exerted by fetal fibroblasts. We next investigated the effect of growth factor treatment (using EGF and PDGF) on adult fibroblast contractility as reflected in the applied traction force. We then examined whether reduction of CCT-eta or CCT-beta via siRNA transfection would affect the contractility of fibroblast cells, either at baseline or in response to growth factor (PDGF). For experiments involving siRNA, cells were transfected (as described above) with CCT-eta and CCT-beta siRNAs and were also co-transfected with the plasmid pdsRed2-C1 (Clontech Laboratories, Inc., Mountain View, CA). This vector contains the red fluorescent protein gene driven by a cytomegalovirus (CMV) promoter, known to be widely expressed in a variety of cell types. After 24 h transfection the cells were switched to quiescent media (RPMI 1640 medium containing 0.1% dialyzed FBS along with antibiotic/antimycotic solution) and left for another 24 h at 37C. Then, 48 h after transfection, fibroblasts were trypsinized off their tissue culture vessels and were placed on the above-described collagen-coated polyacrylamide gel disks at 3000 cells/disk +/2 PDGF (200 nM) and allowed to spread on the collagen surface gel for 6 h in quiescent medium at 37C. Cells visibly expressing red fluorescence after this time were selected (at least 5 cells per disk); CCT-eta & Fibroblast Phenotype these cells are proven to be successfully transfected with pdsRed2- C1, and are therefore highly likely to have taken up siRNA as well. This allowed us to collect traction force measurements only from cells that were likely exhibiting an siRNA mediated downregula- tion of the CCT-eta/CCT-beta subunits, rather than relying on capturing a sufficient number of cells by surveying a large group, which would necessarily include many non-transfected cells. First, phase contrast images of individual cells as well as red fluorescent images were taken, followed by imaging of the underlying green fluorescent beads. Then, the cells on the gel disk were trypsinized (removing the applied contractile force) and images of the fluorescent beads at the same view and the same z plane were taken. An image correlation method was used to compute the bead displacement field by comparing the bead image under the contracting cells with the cell-free bead image. At the beginning of the image processing, the pair of images was corrected for relative translational shift. Then the images were divided into small windows (16 by 16 pixels), and the normalized cross-correlation function was implemented for each window to find the relative displacement for each window between these two images. A hierarchical algorithm, polynomial fitting, and low-pass filtering was used to minimize the noise level. The traction field was calculated from the displacement field by using Fourier transform traction cytometry (FTTC) [36]. The calculation of traction forces is based on the Boussinesq solution for the displacement field on a surface of a semi-infinite solid [37]. At least 20 cells in each experimental group were used to determine their collective average traction forces. Statistical Analyses Statistical analyses were performed by a paired two-tailed Student’s t-test. A p value of ,0.05 was considered significant. Results CCT-eta mRNA and protein levels are reduced in fetal fibroblasts when compared to adult fibroblasts We first examined both CCT-eta and CCT-beta message and protein levels in primary cultures of fetal and adult fibroblasts. qRT-PCR indicated that CCT-eta mRNA expression was significantly elevated in adult fibroblasts (by , seven- fold) when compared to fetal fibroblasts (Figure 1a); in contrast, there was little difference in CCT-beta messenger abundance between fetal and adult fibroblasts (Figure 1b). Immunoblotting demonstrated that CCT-eta protein expression was similarly substantially elevated in adult fibroblasts compared to fetal fibroblasts, whereas CCT-beta protein expression showed no difference between the two cell types (Figures 1c and 1d). Cell migration of fetal and adult fibroblasts is differentially modulated by exogenous growth factors Since one of the main phenotypic attributes of fibroblastic cells relevant to their role in wound healing and scar formation is their ability to migrate into a wound bed, we investigated the migration profiles of fetal and adult fibroblasts in a well- established in vitro assay [35], both at baseline and in response to several growth factors known to be important in the adult wound healing process (EGF and PDGF). Fetal and adult fibroblasts were noted to have comparable migratory profiles at baseline, but, as shown in Figure 2, fetal fibroblasts were completely insensitive to the stimulation of cell migration seen in adult fibroblasts in response to EGF and PDGF. Whereas adult fibroblasts significantly increased their migration rates in PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10063