CCT-eta & Fibroblast Phenotype Figure 2. Cell migration of adult but not fetal fibroblasts is responsive to EGF and PDGF induction. Primary cultures of fibroblasts obtained from fetal and adult rabbit skin were tested for motility in an in vitro wound healing assay. Cells were treated with increasing concentrations of EGF (1 nM and 10 nM) and PDGF (40 nM, 100 nM, 200 nM). The values are normalized to baseline motility and shown as EGF- and PDGF-induced cell motility at each concentration. Fetal and adult fibroblasts had essentially identical baseline motility, but only adult cells responded to growth factor stimulation. The values are mean 6 SEM of six independent studies each performed in triplicate. Statistical analyses were performed by Student’s t-test. doi:10.1371/journal.pone.0010063.g002 siRNA against CCT-eta but not CCT-beta inhibits EGF- and PDGF-induced cell migration in adult fibroblasts Since we have confirmed that CCT-eta is specifically decreased during fetal wound healing, and that CCT-beta is not differentially expressed in either fetal or adult wounds, we were interested to explore the functional significance of the specific inhibition of these target genes, CCT-eta and CCT-beta, on cellular characteristics important to wound healing and scar formation. We therefore investigated what effect the downreg- ulation of CCT-eta and CCT-beta by siRNA in adult fibroblasts would have on their ability to migrate in an in vitro wound healing assay. Near confluent cultures of adult fibroblasts were transfected with siRNA versus CCT-eta (or CCT-beta) or control, then a zone of ‘‘wounding’’ was created by mechanically removing cells with a rubber policeman. Cultures were then stimulated with growth factors (or control vehicle only) and incubated for 48 h while cells moved to repopulate the denuded ‘‘zones of injury.’’ The distance the cells migrated from the acellular front edge was photographed and measured with computer assistance, thus arriving at a quantitative value reflecting cellular motility. The results of this in vitro wound healing assay, which measures the averaged and directional migration of a cell population, were striking and consistent: siRNA-mediated reduction of CCT-eta markedly decreased basal cell motility to 76%66% (p,0.04) compared to no treatment and decreased epidermal growth factor (EGF)-induced cell motility to 108%68% (p,0.01) compared to EGF treatment alone (144%611%, p,0.02) (Figure 4a). In contrast downregulation of CCT-beta with siRNA did not result in any inhibition of either basal cell motility or EGF-induced cell motility (Figure 4b). In no case did scrambled siRNA have any effect on cellular migration when compared to untreated controls. Virtually identical results were obtained when examining the effect of CCT subunit downregulation on cell migration in response to PDGF. Once again downregulation of CCT-eta inhibited both basal cell motility (70%62%, p,0.009 compared to no treatment) and PDGF-induced cell migration (to 105%67%, p,0.01) compared to PDGF treatment alone (151%611%, p,0.05). As in our experiments with EGF, downregulation of CCT-beta did not have any effect on either basal or PDGF- induced cell migration (Figures 5a & 5b). In each of these experiments we confirmed the successful donwregulation of CCT-eta (or CCT-beta) mRNA and protein levels by qRT-PCR and Western blot (as presented in Figure 3) (data not shown). This evidence strongly supports an important and specific role for CCT-eta in fibroblast physiology and reinforces its likely importance to wound healing. Traction force exerted by adult fibroblasts is higher than fetal fibroblasts Having observed that modulation of CCT-eta can affect fibroblast motility, we also sought to measure its effect on cellular contractility. Since one of the major differentiating characteristics of fetal versus adult wound repair is that the latter proceeds with a contraction of the wound bed, we hypothesized that fetal fibroblasts may be inherently less contractile than adult. We therefore first compared the traction forces exerted on their substrata by fetal fibroblasts versus adult fibroblasts using traction force microscopy. Under basal conditions adult fibroblasts apply a significantly stronger traction force (136%629% p,0.04) to their microenvironment than do fetal fibroblasts (Figure 6a). Adult fibroblast contractility is enhanced by stimulation with growth factors We next investigated what effect EGF and PDGF have on adult fibroblast contractility. Cultures of adult fibroblasts were incubated with these growth factors (or control vehicle) and the ability of their component cells to exert traction on their substrata was again quantified by traction force microscopy. Interestingly, although both agents markedly increased cellular contractility, we found that PDGF stimulated cellular traction force (201%615% p,0.03) more highly than EGF (169%69%) (Figure 6b). Since traction force microscopy is a highly labor intensive assay, this observation led us to select PDGF as the stimulator to use in further traction force experimentation. PLoS ONE | www.plosone.org 6 April 2010 | Volume 5 | Issue 4 | e10063