Figure 1. The expression of P311 in different cell types. Total RNA was isolated from cells and RT-PCR was performed to detect P311. The groups were designed as Sc (hypertrophic scar-derived fibroblast- like cells), Sk (skin-derived fibroblasts), Ep (epidermal cells), PBM (peripheral blood monocytes) and mMSC (bone marrow mesenchymal stem cells). Representative result is from 1 of 10 experiments. doi:10.1371/journal.pone.0009995.g001 CTGCCGTACGCGGAATACTTCAGAAAAAACAGCTGTT- CGA-59. These shRNA fragments were ligated with pGenesil-1 plasmid (Genesil, China). Primary human fibroblasts containing about 26104 cells were transfected with the 1 mg P311 shRNA plasmids using 3 ml FuGene 6 non-liposome transfection reagent (Roche, USA). Stable fibroblasts clones were selected with medium containing G418 (400 mg/ml) for 3 weeks and the expression levels of P311 protein were determined by Western blotting. Clones displaying satisfactory P311 gene suppression were selected for subsequent analysis. Scar fibroblasts expressing shRNA gene were divided into the following groups: 1. Sc (hypertrophic scar-derived The Role of Human P311 Gene fibroblast-like cells); 2. control (hypertrophic scar-derived fibro- blast-like cells transfected with negative plasmid, served as control group); 3. shRNA1 (hypertrophic scar-derived fibroblast-like cells transfected with shRNA1 plasmid); 4. shRNA2 (hypertrophic scar- derived fibroblast-like cells transfected with shRNA2 plasmid); 5. shRNA3 (hypertrophic scar-derived fibroblast-like cells transfected with shRNA3 plasmid). Western Blotting Cells were lysed using 1% protease inhibitor cocktail, 1% phenymethylsulphonyl fluoride and 1% sodium orthovanadate in RIPA lysis buffer (Santa, USA). The samples were scraped, collected and centrifuged at 10,000 g for 30 minutes. The supernatant was collected, and protein concentrations were determined by Bradford assay. Equal amounts of protein were mixed with reducing SDS sample buffer and boiled for 5 minutes before loading onto 10% SDS-PAGE gels. Electrophoresis was carried out under reducing conditions at 100 volts for 1.5 hours and the separated proteins were then transferred at 100 volts over 1.5 hours to a nitrocellulose (NC) membrane (GE, USA). The membrane was blocked with Tris-buffered saline (TBS) containing 5% non-fat powdered milk for 2 hours at room temperature and then incubated with rabbit anti-human P311 antibody (1:1000) (Boster, China) at 4uC overnight. The blots were subsequently washed with TBS containing 1% Tween 20 for 4 times and then Figure 2. The effect of P311 on fibroblast functions after P311 gene upregulation. Total RNA was isolated from fibroblasts and RT-PCR was performed to compare a-SMA, TGF-b1 and a1(I) collagen gene(COL1A1) mRNA levels in different treated groups. (A) Hypertrophic scar-derived fibroblast-like cells were transduced with adenovirus P311 and cultured as described under materials and methods. The groups were designed as Sc (hypertrophic 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 +P311). (B) Normal skin-derived fibroblasts were transduced with adenovirus P311. The groups were Sk (normal skin-derived fibroblasts), Sk+Ad (normal skin-derived fibroblasts+adenovirus void vector) and Sk+P311 (normal skin-derived fibroblasts+P311), * means P,0.01 between the two groups. (C, D) Transduction of P311 into normal skin-derived fibroblasts increased the S- Phase cell fraction as determined by FACS. (C) Control group (6200). (D) P311 transduced group (6200). A representative analysis of 3 experiments is shown. doi:10.1371/journal.pone.0009995.g002 PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e9995