tions. Each experiment included controls, which contained the transfection reagent and control siRNA. The transfected cells were subjected to luciferase reporter gene assays, RT-PCR and western blot analysis 48 h after the transfection. Treatment of tumor cells HepG2-X cells were cultured in serum-free medium for 12 h. Briefly, the engineered cells were treated with MK886 (5, 10 or 20 mM), Indo (10, 20 or 50 mM) and PDTC (20, 40 or 60 mM) for 6 h. The treated cells were used to perform luciferase reporter gene assays. The examination of cytotoxicity mediated by MK886 and Indo has been performed [35]. Transfection Transfection was performed in cells using Lipofectamine 2000 (Invitrogen, USA). In brief, the cells were plated in 6-well or 96- well plates at 50% confluence. For each well, siRNA was added into 250 ml Opti-MEM medium (Gibco), and 5 ml Lipofectamine 2000 was added into 250 ml Opti-MEM medium and mixed well. The mixture was added and incubated for 6 h before the medium was replaced. Luciferase reporter gene assays Cells (26105) were plated in a 24-well culture plate and transfected with 300 ng reporter plasmid with 50 ng of pRL-TK encoding renilla luciferase. The treated cells were harvested after 48 h and lysed in 16 passive lysis buffer. The luciferase activity was determined using Dual-Luciferase ReporterH Assay System (Promega, USA) on a Luminometer (TD-20/20, Turner Designs) according to the manufacturer’s instructions. Promoter activity was analyzed by detecting firefly luciferase activity and normal- izing to renilla luciferase activity in each well, as previously described [41]. Each assay was performed in triplicate. RNA extraction, RT-PCR and real-time PCR Total RNA extraction and reverse transcription were carried out as described previously [45]. Synthesized cDNA was used as a template for PCR (primers are listed in table S1). The RT-PCR products were verified by electrophoresis on a 1% ethidium bromide-stained agarose gel. Quantitative real-time PCR was performed using the SYBRH Premix Ex TaqTM II PCR kit (Takara, Japan) by following the manufacturer’s instructions. The relative amounts of mRNAs were calculated using the DDCt method [46] with GAPDH as the endogenous reference gene amplified from the samples. All experiments were performed in triplicate. Western blot analysis After indicated treatments, the cells were washed three times with ice-cold phosphate-buffered saline (PBS). The cytosolic extracts were extracted with lysis buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and protease inhibitor cocktail). Nuclear extracts were collected using NEPER Nuclear and Cytoplasmic Extraction kit (Pierce Biotech- nology, USA). The western blot protocol was described previously [35]. The primary antibodies used were against b-actin (1:1000 dilution, Sigma-Aldrich, USA), HBx (1:1000 dilution, Abcam, UK),Capn4 (1:1000 dilution, Thermo Fisher Scientific, USA), OPN (1:800 dilution, provided by Dr. Chao Bian from Shanghai institutes for biological sciences, Chinese Academy of Sciences), 5- LOX (Santa Cruz Biotechnology, USA),histone-3 (Cell Signaling Technology, USA) and NF-kB/p65 (1:400 dilution, Santa Cruz Biotechnology, USA). All experiments were performed in triplicate. Signal Pathway of Cell Migration Mediated by HBx Enzyme-linked immunosorbent assay (ELISA) The amount of LTB4 (a metabolite of 5-LOX) was examined by ELISA according to the manufacturer’s instructions. The concentration of LTB4 was normalized to the total protein. The protein concentrations in these extracts were determined by a standard protein assay method (Bio-Rad Laboratories, Inc., USA). All experiments were performed in triplicate. Statistical analysis All values are presented as means 6 SEM. Each value is the mean of at least three separate experiments in each group. Data were analyzed by comparing two groups using Student’s t test. P,0.05 was considered significant. Supporting Information Figure S1 HBx upregulates the expression of OPN. (A) The promoter activity of OPN was examined by luciferase reporter gene assay in HepG2-X (or H7402-X) cells, which was abolished by RNAi targeting HBx mRNA, using the indicated doses of pSilencer3.0-X (pSi-HBx) plasmid (**P,0.01 Student’s t test). (B) The protein expression of OPN was detected in HepG2-X (or H7402-X) cells by western blot analysis, which was attenuated by pSi-HBx plasmid in a dose-dependent manner. (TIF) Figure S2 HBx upregulates the expression of 5-LOX. (A) The expression of 5-LOX was examined by real-time PCR in HepG2-X (or H7402-X) cells, which was abolished by using pSilencer3.0-X plasmid (***P,0.001, Student’s t test). (B) The expression level of 5-LOX and HBx were detected by western blot analysis. (C,D) The level of LTB4, a metabolite of 5-LOX, was determined by ELISA in conditioned media or in cell lysates from HepG2-X (or H7402-X) cells (*P,0.05, **P,0.01, ***P,0.00l, Student’s t test). (TIF) Figure S3 HBx upregulates NF-kB, 5-LOX, OPN and Capn4 in HepG2.2.15 cells. The expression levels of NF-kB, 5- LOX, OPN, Capn4 and HBx were detected by western blot analysis. (TIF) Figure S4 HBx increases the promoter activity of OPN and Capn4, but not by HBc and HBs. (A) The promoter activity of OPN was examined by luciferase reporter gene assay in HepG2 cells (**P,0.01 Student’s t test). (B) The promoter activity of Capn4 was examined by luciferase reporter gene assay in HepG2 cells (**P,0.01 Student’s t test). (TIF) Figure S5 HBx promotes hepatoma cell migration through OPN and Capn4, but not by HBc and HBs. (A) The migration ability of hepatoma cells was examined by wound healing assay when the cells were treated by pSi-HBx, Si-OPN or Si-Capn4. Black arrows indicate the wound edge closure of monolayer cells. (B) The average migration distances of the wound edge were measured in three independent experiments (*P,0.05, **P,0.01, Student’s t test). (C) The migration ability of hepatoma cells was examined by wound healing assay when HBx, HBs, HBc, OPN and Capn4 were overexpressed in the cells. Black arrows indicate the wound edge closure of monolayer cells. (D) The average migration distances of the wound edge were measured in three independent experiments (*P,0.05, **P,0.01, Student’s t test). (TIF) Figure S6 HBx promotes cell migration through NF-kB. (A) The migration ability of hepatoma cells was examined by wound PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31458