CCT-eta & Fibroblast Phenotype Figure 1. CCT-eta but not CCT-beta protein and mRNA are differentially expressed in fetal versus adult fibroblasts. RNA and protein extracted from fetal and adult fibroblasts were subjected to qRT PCR (a & b) and Western blot (c & d) analyses respectively. CCT-eta mRNA was significantly more abundant in adult fibroblasts when compared to fetal fibroblasts (a); there was no significant difference in CCT-beta message levels between fetal and adult fibroblasts (b). 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 CCT-eta protein(c). In contrast CCT-beta protein levels were not different between fetal and adult fibroblasts (d). Blots shown here are representative of at least three different experiments. doi:10.1371/journal.pone.0010063.g001 response to several concentrations of these growth factors, fetal fibroblast migration was unaffected. Although many cellular and molecular components are involved in cell migration, this striking observation led us to query whether selective reduction of the CCT-eta subunit alone might alter the migratory abilities of adult fibroblasts, in effect rendering them more ‘‘fetal-like’’ in behavior. siRNA against CCT-eta and CCT-beta effectively and specifically downregulates target mRNA and protein in rabbit adult fibroblasts We employed siRNA technology to experimentally decrease the amount of CCT-eta and CCT-beta message (and protein) in cultures of adult fibroblasts. Cells were transfected either with experimental siRNAs (against actual target sequence) or with scrambled control siRNAs (with identical base pair compositions). After 48 h transfected cells were harvested and analyzed by qRT- PCR and immunoblotting. siRNA versus CCT-eta reduced mRNA levels to 29% of control; this reduction was effective even in the presence of EGF (Figure 3a) (similar results were obtained with PDGF- data not shown). Scrambled siRNA sequence had no effect on CCT-eta message. Neither CCT-beta nor CCT-alpha message levels were affected by siRNA targeted against CCT-eta (data not shown). Importantly, CCT-eta siRNA also significantly reduced CCT-eta protein levels as measured by Western blot (Figure 3c); again, this reduction was effective even in the presence of added growth factors, and control scrambled siRNA elicited no such decrease in target protein. siRNA targeted against CCT-beta was similarly effective, reducing CCT-beta message by some 50% (by qRT-PCR) and CCT-beta protein by greater than 50% (by Western blot) (Figures 3b and 3d). As with CCT-eta, this reduction held even in the presence of growth factors, and control scrambled siRNA sequence had no effect on either mRNA or protein levels. CCT- beta siRNA had no effect on CCT-eta or CCT-alpha message levels (data not shown). PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e10063