P311 expression on the properties of scar-derived fibroblast-like cells. Transfection of scar-derived fibroblast-like cells with a shRNA targeting P311 (shRNA1) significantly lowered the expression levels of P311 protein as detected by western blot (Fig. 4A). These cells displayed reduced levels of TGF-b1 expression (Fig. 4B). Significantly, there was a partial loss of the contractile ability of fibroblast-like cells in the FPCL model following the suppression of P311 (Fig. 4C). Discussion The formation of hypertrophic scar is an abnormal wound healing response after trauma. Histologically and clinically, hypertrophic scar is characterized by excessive fibroblast hypertrophy, accumulation of extracellular matrix and con- tracture [16,17,18,19]. At the molecular level, TGF-b is considered to be the critical regulator involved in the formation of hypertrophic scar [20,21]. However, there are currently no approaches based on this paradigm which completely prevent scar formation or treat existing scar [1,20,22]. This leads us to search for additional potentially unknown factors involved in hypertrophic scar formation, which might aid our understanding of hypertrophic scar The Role of Human P311 Gene formation. Such information might also suggest novel strate- gies for controlling scar formation. Our previous microarray analysis revealed that P311 was highly upregulated in hypertrophic scar tissue [3]. Meanwhile, Pan et. al. showed that P311 was found in human wounds during healing and disappeared after wound healed [7]. Importantly, Pan also demonstrated that transfection of P311 into murine cell line NIH 3T3 or C3H10 T1/2 could significantly stimulate the differentiation of fibroblasts to myofibroblasts via a TGF-b1 independent signaling pathway. Significantly, these results were not obtained with human fibroblasts. The combination of our previous work [3] and that of Pan et. al. prompted us to explore the possible roles of P311 in human fibroblast function. An examination of P311 mRNA expression in a number of human cell types revealed that P311 mRNA expression was only detected in hypertrophic scar-derived fibroblast-like cells. These results are consistent with the finding of immunohistochemical studies of Pan et. al. [7]. These observations suggest that P311 expression may be involved in normal and hypertrophic scar formation. Our results show that the expression of P311 can induce the expression of a–SMA (Fig. 2B) in normal skin-derived fibroblasts. As a-SMA is a putative cell marker of myofibroblasts [23], this result suggests that P311 may induce the myofibroblast phenotype. Figure 4. The expression of TGF-b1 by fibroblasts after P311 shRNA plasmid transfection. (A) Total protein was extracted from hypertrophic scar-derived fibroblast-like cells transfected with P311 shRNA plasmids. Western blotting demonstrated that shRNA1 plasmid was the most effective plasmid for silencing P311 protein expression in fibroblasts. * means P,0.01 between the two groups. (A1) The expression of P311 protein is demonstrated by Western blotting. (A2) The lowest expression of P311 protein was found in shRNA1 group. (B) Hypertrophic scar-derived fibroblast-like cells transfected with P311 shRNA plasmids showed lower expression of TGF-b1 than control. * means P,0.01 between the two groups. (B1) Total RNA was isolated from hypertrophic scar-derived fibroblast-like cells and RT-PCR was performed to determine TGF-b1 mRNA level. (B2) Histogram showed the downregulation of TGF-b1 mRNA level in P311 silenced hypertrophic scar-derived fibroblast-like cells. (C) Hypertrophic scar-derived fibroblast-like cells were transduced with shRNA of P311. The groups were Sc+control (hypertrophic scar-derived fibroblast-like cells + negtive vector (control)) and Sc+ shRNA1 (hypertrophic scar-derived fibroblast-like cells + P311 shRNA1). (C1) Hypertrophic scar-derived fibroblast-like cells were transfected with P311 shRNA plasmids and cultured in a collagen lattice for 2 weeks. (C2) Absence of P311 inhibited the contractile ability of hypertrophic scar-derived fibroblasts. * means p,0.01 considered as statistical significance as compared with the control group with negative shRNA plasmids (control). A representative analysis of 3 experiments is shown. doi:10.1371/journal.pone.0009995.g004 PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e9995