Immunodetection was performed using anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA), anti-α-tubulin (Sigma-Aldrich, St. Louis, MO), and anti-C-Jun (BD Biosciences, Franklin Lakes, NJ) Abs. HepG2 cells were transfected with different combinations of plasmids using FuGENE 6 reagent (Roche, Indianapolis, IN), according to the manufacturer’s protocol. Plasmids used included Myc/pcDNA3.1+ vector containing various forms of HBx, MMP10-WT/pGL3-Basic, MMP10-AP1-Mut/pGL3-Basic AZD1152 HQPA reporter constructs, and an internal control (pRL-SV40). The total amount of expression vectors was equalized with the empty vector. Twenty-four hours after transfection, luciferase and

Renilla luciferase activities were measured by the Dual Luciferase Reporter assay system (Promega, Madison, WI), according to the manufacturer’s protocol. Transfection efficiency was normalized with the Renilla luciferase activity. Experiments were done thrice Ku 0059436 independently. Cells (3 × 106) were seeded 1 day before harvest and chromatin immunoprecipitation (ChIP) assay was performed. Cells were fixed with 1% formaldehyde for 10 minutes, and the reaction was neutralized by adding glycine to a final concentration of 125 mM in the mixture. Formaldehyde cross-linked

cells were collected by centrifugation, resuspended in membrane containing lysis buffer (5 mM of KOH [pH 8.0], 85 mM of KCL, 0.5% NP-40, 0.5% SDS, and 1×CompleteProtease Inhibitors), and incubated on ice for 30 minutes. Cell nuclei were collected by

centrifugation, and cross-linked DNA was digested by Micrococcal nuclease for 20 minutes, according to manufacturer’s protocol (New England Biolabs, Inc., Ipswich, MA). Digested DNA was released from nuclei by freeze-thaw MCE cycles and processed for ChIP assay according to the EZ-Chip assay kit (Millipore, Billerica, MA) protocol. The Ab against C-Jun protein was used (Santa Cruz Biotechnology), and the primer set (forward 5′-CAAACACAGAAATCATTTCCTGG-3′ and reverse 5′-AGATCACCAACAGTATGATTCATGC-3′) covering the putative AP-1-binding site on the MMP10 promoter was employed for standard PCR measurement in the ChIP assay. Clinicopathological features of HCC patients, including tumor size, cellular differentiation according to Edmondson’s grading, venous invasion into portal or hepatic venules, direct liver invasion, tumor microsatellite formation, tumor encapsulation, and number of tumor nodules, were analyzed using PASW Statistics 18 for Windows (SPSS, Inc., Chicago, IL). For clinicopathological correlation analysis, Fisher’s exact test was used for analysis of categorical data. For in vitro cell-invasion assay and reporter assay, the Student t test was used for continuous data. Results were considered significant if the P value was less than 0.05.