CCT-eta & Fibroblast Phenotype Figure 6. Adult fibroblasts are more contractile than fetal fibroblasts. (a) Fetal fibroblasts are less contractile than adult fibroblasts as determined by traction force microscopy. Each bar represents mean 6 SEM of more than 20 cells from two independent experiments. Statistical analyses were performed using Student’s t test. (b) PDGF treatment of adult fibroblasts results in an increase in the observed cumulative traction force; EGF treatment results in a similar although smaller increase. Each bar represents mean 6 SEM of more than 25 cells from two different experiments. Statistical analyses were performed using Student’s t test. doi:10.1371/journal.pone.0010063.g006 we first synthesized an siRNA specifically targeting rabbit a-SMA. Adult fibroblasts were transfected either with experimental siRNA (against actual target sequence) or with a non-specific control siRNA. After 48 h the transfected cells were harvested and analyzed by qRT-PCR and immunoblotting. siRNA versus a-SMA reduced mRNA levels to ,50% of control; this reduction was effective even in the presence of EGF (Figure 10a). The non-specific siRNA control had no effect on a- SMA message. Importantly, a-SMA siRNA also significantly reduced a-SMA protein levels as measured by Western blot (Figure 10b); again, this reduction was effective even in the presence of added growth factor, and non-specific siRNA control elicited no such decrease in target protein. No effect on cellular beta-actin was seen with either siRNA construct (data not shown). Figure 7. siRNA against CCT-eta but not CCT-beta reduces PDGF-induced cellular traction force in adult fibroblasts. Adult fibroblasts transfected with CCT-eta (a) or CCT-beta siRNA (b) along with pDSRed2-C1 were quantified for microdisplacement fields of red fluorescent cells on the green fluorescent substrate. Each assay was repeated twice with more than 30 cells quantified in each experiment. CCT-eta siRNA abolished the increased cellular traction force seen with PDGF treatment (200 nM), whereas CCT-beta siRNA and scrambled controls did not. Values are means 6 SEM of two independent experiments with statistical analyses performed using Student’s t test. doi:10.1371/journal.pone.0010063.g007 PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10063