392 Exp. Mol. Med. Vol. 44(6), 387-393, 2012 down, the supernatant was incubated with 1 μg specific an- tibody (anti-galectin-3, anti-c-Jun and anti-Fra-1, respectively) overnight at 4oC, followed by incubation with 40 μl pro- tein-A/G agarose beads for 1 h. Mouse/rabbit IgG (Santa Cruz) was used as the negative control. After the in- cubation, beads were washed 3 times in RIPA buffer be- fore being dissolved in SDS-PAGE loading buffer. Western blot analysis was performed as described above. Transfilter migration and invasion assays Transfilter migration and invasion assays were carried out with 8.0-μm pore inserts in 24-well Transwells (Corning Costar). Briefly, B16F10 cells were transfected with ga- lectin-3 or MMP-1 siRNAs. One day after the transfection, cells were isolated and added to upper Transwell cham- bers with 0.5 mg/ml collagen type I (BD Bioscience) coated filters for the migration assay, and with 1/15 dilution of ma- trigel (BD Bioscience) coated filters for the invasion assays. DMEM containing 10% FBS and 1% antibiotics was added to the lower chamber and incubation continued for 20 h. Cells that had migrated or invaded into the lower chamber were quantified after H&E staining. For quantification, cells on were counted at 5 randomly selected areas in each well using a wide-field microscopy. Data were expressed as mean ± SD from three independent experiments. Wound healing assays B16F10 cells were seeded into 6-well culture dishes at 1 × 105 cells/well, and maintained in DMEM containing 5% FBS for 24 h. The nearly confluent cells were scratched with a pipette tip and cellular debris was removed. The scratched monolayer was then incubated for another 24 h in DMEM. Cells migrating into the gap were visualized and counted under a microscope. Lentivial galectin-3 shRNA construction and stable cell line establishment Lenti-viral vectors containing galectin-3 shRNA (Gal-3shRNA) was constructed by inserting the double strand gal-3 shRNA into the lentiviral vector pLL3.7 as described pre- viously (Kim et al., 2010). The sequence of the shRNA was as following: gal-3shRNA sense: 5'-TGAACAACAGGAGA GTCATTGTTTCAAGAGAACAATGACTCTCCTGTTGTTC TTTTTTC-3'; gal-3shRNA antisense: 5'-TCGAGAAAAAA GAACAACAGGAGAGTCATTGTTCTCTTGAAACAATGAC TCTCCTGTTGTTCA-3'. The lentiviral vector containing lacZ was used as a control. Lentiviral infection of B16F10 melanoma cells was performed as described previously (Kim et al., 2010). Approximately 80% cells were infected and confirmed with FACS analysis. Mouse pulmonary metastasis model Female C57BL/6 mice, purchased from Orient Bio in Korea, were maintained in the animal facility at the National Cancer Center (NCC). B16F10 cells (1 × 106 cells/ml in 100 μl PBS) infected with lentiviral lacZ (control) or gal-3shRNA was injected into 6-week-old female C57BL/6 mice (five mice per group) through the lateral tail vein. Four weeks after the injection, mice were sacrificed with an overdose of anesthesia. The number of metastatic nodules on the surface of the lung was counted under the surgical microscope. Chromatin immunoprecipitation assay A chromatin immunoprecipitation (ChIP) assay was carried out using a ChIP assay kit (Upstate). Samples were applied to dishes after galectin-3 siRNA treatment and assays were conducted following the manufacturer’s instructions. Anti-galectin-3, c-Jun and Fra-1 and normal mouse/rabbit IgG were used to immunoprecipitate DNA containing complexes. Prior to PCR, primers were prepared for AP-1 with MMP-1 promoter binding sites: MMP-1 promoter (-2950) 5'-AAGAAGAAGGTGGCCAGGAT-3' and (-2901) 5'-TGCCTTCATTT TCCATTTCC-3'. PCR was performed with Ex Taq (Takara). Cell proliferation assay B16F10 cells, growing in 96-well culture plates at a density of 3 × 103 cells/well，were transfected with scrambled siRNA, galectin-3 siRNA, and MMP-1 siRNA respectively. 24 or 40-h post transfection, cell viability was analyzed by MTT assay, as described previously (Cheong et al., 2010). Luciferase assays B16F10 melanoma cell lines were co-transfected with an AP-1 reporter plasmid and galectin-3 siRNA 48 h before the luciferase assay was performed, as described pre- viously (Kim et al., 2010). Statistics analysis All data were obtained from at least three independent ex- periments and are presented as mean ± SD, unless other- wise indicated. Statistical analysis was performed using one-way ANOVA. Data were considered significant if P ＜ 0.05. Acknowledgements This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare (A110961), Republic of Korea. References Ahonen M, Baker AH, Kahari VM. Adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells. Cancer Res 1998;58:2310-5 Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1991;1072:129-57