CCT-eta & Fibroblast Phenotype Figure 8. mRNA and protein levels show that a-SMA level is significantly increased in adult fibroblasts in comparison to fetal fibroblasts. RNA and protein extracted from fetal and adult fibroblasts were subjected to qRT PCR (a) and Western blot (b) analyses respectively. The a-SMA mRNA levels were significantly more abundant in adult fibroblasts when compared to fetal fibroblasts (a). Values are means 6 SEM of three independent studies performed in duplicate. Statistical analyses were performed using Student’s t test. NS = non-significant. Equal amounts of protein loaded from fetal and adult fibroblasts showed that adult fibroblasts express significantly greater a-SMA protein (b). GAPDH was used as loading control. doi:10.1371/journal.pone.0010063.g008 siRNA against a-SMA inhibits both basal and EGF- induced cell migration in adult fibroblasts Direct reduction of a-SMA was then accomplished using siRNA against a-SMA in the setting of our in vitro wound healing assay to determine what effect this would have on fibroblast motility. As seen in Figure 11, depletion of a-SMA resulted in both decreased basal cell motility (86%66% p,0.01 compared to no treatment) and in an abrogation of the ability of the cell to respond to the EGF motive stimulus (to 94%64% p,0.001). Control siRNA had no such effects. These results mirror the phenotypic changes effected by the specific depletion of the CCT- eta subunit. Discussion Although the scarless wound healing observed in fetal integumentary tissues has been known for some time, the critical mechanisms that enable tissue repair to occur absent scar formation are still unclear. Since fibroblasts are the end effector cells of scar formation, ultimately such mechanisms must manifest themselves in fibroblast physiology. This report examines two parameters of fibroblast physiology critical to wound healing and scar formation, motility and contractility, and how they are affected by modulation of the CCT subunits eta and beta. cDNAs of CCT-eta and –beta from rabbit confirm that these polypeptides display a significant degree of sequence homology, Figure 9. siRNA downregulation of CCT-eta but not CCT-beta reduces a-SMA protein levels in adult fibroblasts. (a & b)Western blot showed effective reduction of both CCT-eta and a-SMA protein levels when CCT-eta siRNA was administered, leaving beta-actin largely unaffected. In contrast, downregulating CCT-beta did not lead to significant reduction in either a-SMA or beta-actin levels. GAPDH was used as loading internal control. A representative immunoblot of up to three similar such blots are shown for each analysis. Ntx- no transfection; EGF-EGF treatment (1 nM); siRNA-treatment with CCT-eta/CCT-beta siRNA; Scr- treatment with scrambled control siRNA. doi:10.1371/journal.pone.0010063.g009 PLoS ONE | www.plosone.org 10 April 2010 | Volume 5 | Issue 4 | e10063