CCT-eta & Fibroblast Phenotype Figure 4. siRNA against CCT-eta decreases EGF - induced fibroblast migration, whereas siRNA against CCT-beta does not. Cells were incubated in the presence or absence of EGF(1 nM) +/2 siRNA against CCT-eta (a) or CCT-beta (b) in an in vitro wound healing assay. In all experiments a subunit -specific scrambled siRNA sequence was used as control. Cell motility is displayed as a relative percentage of baseline motility in the absence of EGF or siRNA exposure (100%). Active siRNA versus CCT-eta reduced both basal and EGF-induced motility; siRNA versus CCT-beta and scrambled controls had no effect. Values are means 6 SEM of six independent studies, each performed in triplicate. Statistical analyses were performed with Student’s t test. doi:10.1371/journal.pone.0010063.g004 dramatic and specific decrease in a-SMA protein but did not have any effect on cellular beta-actin protein (Figure 9a). In contrast, CCT-beta inhibition showed no significant effect on either a-SMA or beta-actin protein accumulation (Figure 9b). siRNA against a-SMA effectively and specifically downregulates target mRNA and protein in rabbit adult fibroblasts We next wished to examine the effect of direct downregulation of a-SMA on basal and EGF-induced cell migration. To this end Figure 5. siRNA against CCT-eta decreases PDGF-induced fibroblast migration, whereas siRNA against CCT-beta does not. Cells were incubated in the presence or absence of PDGF (200 nM) +/2 siRNA against CCT-eta (a) or CCT-beta (b) in an in vitro wound healing assay. In all experiments a subunit -specific scrambled siRNA sequence was used as control. Cell motility is shown as a percentage of baseline migration in the absence of PDGF or siRNA exposure. As with EGF, active siRNA targeting CCT-eta inhibited basal and PDGF-induced motility, whereas CCT-beta siRNA and scrambled controls did not. Values are means 6 SEM of six independent studies, each performed in triplicate. Statistical analyses were performed with Student’s t test. doi:10.1371/journal.pone.0010063.g005 PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10063