CCT-eta & Fibroblast Phenotype Figure 3. siRNAs against CCT-eta and CCT-beta decrease both basal and EGF- induced mRNA and protein levels of their targets in fibroblasts. (a & b) qRT-PCR analysis of CCT-eta and CCT-beta mRNA levels showed effective inhibition of both basal expression and EGF-induction in siRNA-transfected adult fibroblasts. Results are expressed as relative quotient (RQ) of measured CCT-eta or CCT-beta mRNA and were calculated as a percentage of baseline control levels (100%). Values are means 6 SEM of six independent studies, each performed in duplicate. Statistical analyses were performed with Student’s t test. Ntx- no transfection; EGF-EGF treatment (1 nM); siRNA-treatment with CCT-eta/CCT-beta siRNA; Scr- treatment with scrambled control siRNA. (c & d) Western blot results using CCT-eta and CCT-beta antibody (1:500) showed effective reduction of CCT-eta and CCT-beta protein levels when siRNA was administered but no decrease when scrambled siRNA was employed. GAPDH was used as a loading control. A representative immunoblot of up to four similar such blots is shown for each analysis. doi:10.1371/journal.pone.0010063.g003 siRNA against CCT-eta but not CCT-beta inhibits PDGF- induced traction force in adult fibroblasts We next examined the effect of downregulation of CCT-eta by siRNA treatment on the cellular contractility of adult fibroblasts. Cultures of adult fibroblasts were transfected as described above with CCT-eta/CCT-beta siRNA (or the relevant controls), then stimulated with PDGF treatment. Cultures were simultaneously transfected with a marker vector expressing red fluorescent protein; this allowed us to selectively assay the traction forces of cells known to have taken up exogenous genetic material, maximizing the likelihood that such cells had also incorporated our siRNAs of interest (when present). Our results demonstrate that CCT-eta siRNA, while having no effect on the contractility of unstimulated cells, does abrogate the increased traction/contrac- tility seen with PDGF treatment (108%64%, p,0.01) compared to PDGF treatment alone (163%63%, p,0.02). In contrast, CCT-beta siRNA has no effect on either basal cell contractility or on the PDGF-dependent increase in cellular traction (Figures 7a and 7b). Scrambled siRNAs had no effect on cellular traction in any instance. Our data therefore indicate that, in addition to diminishing the motile response of fibroblast cells, selective downregulation of CCT-eta can also inhibit their ability to contract in response to exogenous agents. This reduced potential contractility may also play a role in discriminating between a fetal and adult wound healing phenotype. a-SMA levels are significantly elevated in adult fibroblasts in comparison to fetal fibroblasts Since chaperonins are known to mediate the folding of cytoskeletal actin, as an initial study we determined the mRNA and protein levels of a-SMA in fetal and adult fibroblasts. We found that both mRNA and protein levels of a-SMA were significantly higher in adult fibroblasts when compared to fetal fibroblasts (Figures 8a and 8b). siRNA against CCT-eta but not CCT-beta markedly reduces a-SMA protein levels but did not affect beta- actin levels in adult fibroblasts We next sought to determine what effect inhibiting CCT-eta and –beta subunits specifically would have on cellular a-SMA expression. We found that inhibiting CCT-eta resulted in a PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e10063