incubated with HRP-labeled goat anti-rabbit secondary antibody (1:2000) (Boster, China) for 2 hours at room temperature. Proteins were washed with TBS containing 1% Tween 20 for 4 times again, and visualized using enhanced chemiluminescence (Pierce, USA) according to the manufacturer’s instructions. RT-PCR P311 mRNA expression was detected in skin- and hypertrophic scar-derived fibroblasts by RT-PCR, and the expression of a- SMA, TGF-b1, a1(I) collagen gene (COL1A1) mRNAs were detected in fibroblasts before and after P311 gene transduction. The primers for the genes were designed according to the sequence registered in Genbank, and synthesized by Shanghai Bioaisa Biotech Co. Ltd. Total RNA was extracted from cultured cells with TRIzol reagent (Gibco, USA). RT-PCR was performed as described previously. The RNA was made at 42uC for 15 minutes using AMV reverse transcriptase (Promega, USA) and oligo(dT) as a primer. The optimal reaction conditions were determined for each experimental group and primers. The primers for P311 were 59-GCACATATGGTTTATTACCCAG- 39 and 59-CAGGATCCTTAAAAAAAGTGGAGG-39. The primers for a-SMA were 59-GGTGATGGTGGGAATGG-39 and 59-TGGCTGGAACAGGGTCT-39. The primers for TGF- b1 were 59-CTGTGGCTACTGGTGCTGAC-39 and 59-CAT- AGATTTCGTTGTGGGTTTC-39. The primers for a1(I) colla- gen gene(COL1A1) were 59-TCTTTGAATCCTAGCCCATC- 39 and 59-GGAGCACCTTTACAAGCAGT-39. The primers for glyceraldehyde phosphodehydrogenase(GAPDH) were. 59-CG- TCTTCACCACCATGGAGA-39 and 59-CGGCCATCGCCA- CAGTTT-39. Reaction conditions were 5minutes at 94uC and 35 cycles of 94uC for 30 seconds, 55uC for 30 seconds, and 72uC for 40 seconds. GAPDH was used as an internal control. PCR products were separated by electrophoresis on 1% agarose gels, stained with ethidium bromide, and photographed against ultraviolet light. Expression levels of target genes were normalized by concurrently measured GAPDH mRNA levels. The mRNA relative content can be showed by the ratio of its PCR and GADPH absorbency. Analysis of cell cycle by FACS Cells were detached in 0.25 g/l trypsin and washed twice in ice- cold PBS. Followed by fixation in 70% ethanol in 4uC overnight, cells were washed 2 additional times in PBS, and then stained for 30 minutes at 37uC in 50 mg/ml propidium iodide (Sigma, USA) solution containing 200 mg/ml RNase A and 0.1% Triton-X-100. Samples were stored at 4uC until analysis by FACS (BD, USA). Statistical analysis Measurement data were presented as means 6 standard deviation (SD) and analyzed by one-way ANOVA test in conjunction with LSD test. The differences between multiple specimens were analyzed when necessary. P value less than 0.05 was considered as the statistical significance. Results The distribution of P311 The expression pattern of P311 mRNA was examined in several human cell types by means of RT-PCR. P311 was readily detectable in dermal fibroblast-like cells derived from hypertrophic scar tissue. However, it was not detected in the normal skin- derived dermal fibroblasts, epidermal cells, peripheral blood mononuclear cells or bone marrow mesenchymal stem cells (Fig. 1). The Role of Human P311 Gene The effect of P311 over expression on fibroblast functions The effects of transduction of the human P311 gene or its empty control virus into hypertrophic scar-derived or normal dermal fibroblasts were examined. We observed that normal skin-derived fibroblasts do not express a-SMA or TGF-b1 (Fig. 2B). Following transduction with a P311 expression vector, a-SMA, TGF-b1, and COL1A1 mRNAs were all increased (Fig. 2B). Moreover, the proportion of cells in S phase was increased from 0.3% to 6.0% following P311 expression (Fig. 2C and 2D). In contrast hypertrophic scar-derived dermal fibroblast-like cells constitutively express a-SMA, TGF-b1, and COL1A1 (Fig. 2A1 and 2A2). However, the upregulation of P311 did increased the expression of a-SMA, TGF-b1, and COL1A1 mRNA. The ability to induce collagen contraction is a characteristic feature of myofibroblasts [15]. A comparison of the contractile activities of normal skin-derived fibroblasts with those derived from hypertrophic scar indicated that the normal cells expressed lower activity (Fig. 3). Transduction of P311 into normal skin- derived fibroblasts markedly enhanced their ability to contract collagen gels (Fig. 3). Similarly transduction of P311 into hypertrophic scar derived fibroblast-like cells significantly en- hanced their ability to contract the gels (Fig. 3). The suppression of P311 expression inhibited scar- derived fibroblast-like cells functions The above observations suggested that P311 expression induces a number of the phenotypic properties observed in hypertrophic scar derived fibroblast-like cells. Furthermore as P311 expression was elevated in scar tissues, we tested the effects of inhibition of Figure 3. The contraction of collagen by fibroblasts. Normal skin- and hypertrophic scar-derived fibroblasts were transduced with recombinant adenovirus P311 as described in Materials and Methods Section. The groups were designed as Sk (normal skin-derived fibroblasts), Sk+Ad (normal skin-derived fibroblasts+adenovirus void vector), Sk+P311 (normal skin-derived fibroblasts+P311), Sc (hypertro- phic scar-derived fibroblast-like cells), Sc+Ad (hypertrophic scar-derived fibroblast-like cells +adenovirus void vector and Sc+P311 (hypertrophic scar-derived fibroblast-like cells +P31). (A) Normal skin- and hypertro- phic scar-derived fibroblasts were transduced with adenovirus P311, and cultured in a collagen lattice for 13 days. (B) P311 enhanced the contractile ability of fibroblasts obviously. The FPCL model was cultured for 1, 2, 3 and 4 weeks. The contraction index was calculated as previous described. * means p,0.01 as compared with the control group without P311. m means p,0.01 between hypertrophic scar-derived fibroblast-like cells and normal skin-derived fibroblasts. A representative analysis of 3 experiments is shown. doi:10.1371/journal.pone.0009995.g003 PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e9995