390 Exp. Mol. Med. Vol. 44(6), 387-393, 2012 Figure 3. Effect of galectin-3 on MMP-1 expression through binding with AP-1 complex and effect of MMP-1 silencing on the motility of B16F10 mela- noma cells. (A) mRNA and protein expression levels of galectin-3 or MMP-1 were detected by RT-PCR and western blotting respectively after trans- fection of B16F10 melanoma cells with galectin-3 or MMP-1 siRNA. scRNA was used as negative control and β-actin was used as a loading control. (B) Cell proliferation was detected after transfection of B16F10 cells with scrambled siRNA, galectin-3 siRNA or MMP-1 siRNA for 24 and 40 h by MTT assay as described in the Methods section ( *P ＜ 0.001, **P ＜ 0.001, ◦P ＜ 0.001, ◦◦P ＜ 0.001). (C, D) Wound healing assays utiliz- ing B16F10 melanoma cells were performed by scRNA or MMP-1 siRNA transfected cells at 0, 12, 18 and 24 h after creating a scratch. (C) The phase micrographs of B16F10 cells were obtained according to the data. (bar=100 μm) and (D) quantification of cell migration using the monolayer wound healing assay was performed as described in the Methods section. All ex- periments were performed individually in triplicate. (E) Interaction between galectin-3 and AP-1 (Fra-1 and c-Jun) was measured by im- munoprecipitation as previously described in “Methods”, and then ex- pression of galectin-3, Fra-1 and c-Jun was detected by western blot. Whole-cell lysates (WCLs) were used as a positive control. (F) Luciferase activity of AP-1 in lacZ and galectin-3 knock down cells. Luciferase assay was performed using an AP-1 expression luciferase vector transfection to lacZ and galectin-3 knock down cells (P ＜ 0.01, vs. Cont). (G) The prox- imal AP-1 binding site which is located between -80 and -68 from the ini- tiation codon of the MMP-1 gene. (H) A ChIP assay was performed using an- tibodies against galectin-3, c-Jun and Fra-1 in melanoma cells transfected with scRNA and galectin-3 siRNA. In the input lane, total genomic DNA was used as a control for the PCR reaction. Figure 4. Suppressive effects of galectin-3 silencing on metastasis of B16F10 melanoma cells in lung metastasis mouse models. (A) Expression levels of galectin-3 were detected by real time-PCR analysis in B16F10 cells infected with lenti-virus expressing LacZ or galectin-3 shRNA (gal-3shRNA). The preparation and infection of these lenti-vi- ruses were described in the Methods section. (B) Expression levels of galectin-3 and MMP-1 were detected by RT-PCR and western blot analy- sis in B16F10 melanoma cells transfected stably with lacZ and gal-3shRNA respectively. β-actin was used as a loading control. (C, D) Four weeks after tail vein injection of PBS (negative control), parent B16F10 melanoma cells, lacZ expressing B16F10 cells or gal-3shRNA expressing cells into C57BL/6 mice, the formation of lung colonies was counted and illustrated as a histogram (P ＜ 0.01 vs. lacZ group) (C) and photographs (D). The details are described in the Methods section. Discussion This study was performed to better understand the mechanisms underlying the role of galectin-3 in melanoma invasion and metastasis. We have previously shown that after silencing of galectin-3, some genes such as MMP-1 and MMP-3 are down-regulated , while other genes, such as serine protease inhibitor and tissue inhibitors of MMP-2 were up-regulated (data not shown) (Cheong et al., 2010). Among these genes, MMP-1 was down- regulated by galectin-3 to a greater extent than others, which was further confirmed by the following findingsWe confirmed the extensive expression of galectin-3 in different mouse cell lines including B16F1, B16F10 (melanoma), 4T-1 (breast carcinoma), TC-1 (lung cancer) and NIH3T3 (embryonic fibroblast), which may imply its conservation and significance in many types of cancer. However, much higher expression of MMP-1 was detected in the melanoma cell line B16F10 than in the 4T-1,