regulator of avb3 integrin-mediated melanoma cell migration. Our results showed that inhibition of MMP-2 activity in the tumor cells dramatically decreased the adhesion and migration of the tumor cells. Materials and Methods Cell Culture and Proteinase Inhibitors Human A375 melanoma cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Science (Shanghai, China). Human melanoma cell M21 and M21- L were from the School of Basic Medical Sciences, Jilin University of China, and the M21-L is a mutant cell line lacking avb3 integrin [44]. The cells were grown at 37uC in a humidified atmosphere with 5% CO2 in IMDM containing 10% fetal bovine serum (FBS). Serum starvation of cultures was performed as follows: cells were cultured in serum-free medium for 24 h to synchronize the cell cycle at the G0 stage. GM6001 (Chemicon, Temecula, CA) is a broad spectrum hydroxamate MMP inhibitor, and its IC50 values have been reported as follows: 0.4 nM for MMP-1; 0.5 nM for MMP-2; 27 nM for MMP-3; 0.1 nM for MMP-8 and 0.2 nM for MMP-9 [45]. Statistics of the Cell Morphology Cells in the logarithmic phase were trypsinized, re-suspended in IMDM containing 10% FBS, and incubated with MMPs inhibitor (GM6001) at different concentrations (0.4 nM, 0.5 nM, 27 nM, 0.1 nM and 0.2 nM) or with arginine-glycine-aspartate RGD peptides (Sigma, 0.1 mg/ml). The treated cells were seeded in 6- well plates coated with human fibronectin and cultured at 37uC in a humidified atmosphere with 5% CO2. The cell morphology was observed at different time points (3 h, 6 h, 9 h, 12 h, 15 h, and 18 h) using microscopy. Assay of Cell Migration in the Agarose Drop Cell migration was quantified by measuring the extent of cell migrating out of the agarose drops using a modified method [46]. After 24 h of serum starvation, cells were trypsinized and re- suspended at a density of 56106 cells/ml in serum-free IMDM containing 0.3% low melting-point agarose (Invitrogen), and the mixture was maintained at 37uC to prevent the setting of agarose. Two-microliter drops were seeded on fibronectin-coated coverslips in a 24-well plate and then placed at 4uC for 20 min to allow the agarose getting solid. Different concentration of GM6001 was used in this model. Cell migration was measured at 24 h intervals during 3 days under an inverted microscope. The drop area and total area (area of the drop + area of migrating cells) were measured, and cell migration was expressed as the extent of the drop = [(total area/drop area)6100] - 100. The mean values 6 SEM were obtained from at least three independent experiments. Transwell Assay After 24 h of serum starvation, cells were trypsinized and re- suspended in serum-free IMDM at a density of 16105 cells/ml. The cells were seeded onto the top of the insert that was previously coated with fibronectin in serum-free medium containing different agent. Serum was placed in the lower chamber and the cultures were maintained at 37uC in a humidified atmosphere with 5% CO2. Immunofluorescence Assay in the Agarose Drop Model Cells were cultured in the agarose drops on coverslips. The coverslips were fixed in 4% paraformaldehyde and rinsed with phosphate buffer saline (PBS). The cells were pre-incubated with MMP-2 Promotes avb3 Mediated Tumor Cell Migration 5% FBS for 30 min and incubated with the following primary antibodies: mouse anti-human avb3 (1/100, Millipore) and rabbit anti-human MMP-2 (1/100, Santa Cruz, CA). The antibodies were diluted in PBS containing 5% FBS for 1 h before use. The primary antibody-labeled cells were rinsed with PBS, and then incubated with anti-mouse TRITC-conjugated or anti-rabbit FITC-conjugated secondary antibody for 30 min, and then rinsed with PBS. The coverslips were mounted and observed under a confocal microscope (Olympus, Japan). Images were analyzed using the Olympus confocal software. Cell Adhesion Experiment Forty-eight-well plates were coated with 10 mg/ml human fibronectin for 20 min at 37uC. Before addition of cells, the wells were blocked with 0.5% bovine serum albumin (BSA) for 30 min to prevent nonspecific binding. Cells were trypsinized and incubated with calcein AM (It can be transported through the cellular membrane into live cells, which makes it useful for testing cell viability and for short-term labeling of cells) at 37uC for 30 min. The cells were seeded on fibronectin at a density of 16106 cells/ml in serum-free medium, alone or with different concentration of GM6001, for 15 min at 37uC. The adhesion assay was halted by adding a large volume of PBS, and the loosely attached cells were washed off. Fluorescence detection of calcein AM in the plates was conducted using the fluorescence image plate reader (American Molecular Devices. Inc. Gemini EM) with excitation 485 nm and emission 535 nm. MMP-2 Cleavage of Fibronectin Recombinant human MMP-2 (R&D Systems) was activated using p-aminophenylmercuric acetate (APMA) (Merck) for 1 h at 37uC. Full-length fibronectin was cleaved by activated MMP-2 for 2 h at 37uC. One hundred nanograms of MMP-2 and APMA- activated MMP-2 was analyzed by gelatin zymography. MMP-2 cleavage of fibronectin was analyzed using a 10% Tris-HCl gel by silver staining. For the nitrocellulose adhesion assay [47], full- length or MMP-2 cleaved fibronectin was resolved on a native Tris-HCl gel (4%–20%). Proteins were transferred on nitrocellu- lose, and the blot was washed with PBS and incubated with A375 cells for 4 h. After adhesion, the blot was washed, cells were fixed with 10% formaldehyde, stained with 0.1% amido black, and destained with a methanol/acetic acid/water solution (90:2:8). Cells bound to nitrocellulose was shown as dark blue. Results MMP-2 and avb3 Integrin are Expressed in Human A375 Melanoma Cells To study the relationship of MMP-2 and avb3 integrin in tumor cell adhesion and migration, human A375 melanoma cells were suspended or seeded onto coverslips coated with human fibronec- tin. The expression of MMP-2 and avb3 integrin on the tumor cells was evaluated by immunofluorescence method. As shown in Figure 1, MMP-2 and avb3 integrin were expressed on the surface of suspended A375 cells and partially co-localized with each other (Figure 1A). The flow cytometric analysis (FACS) also showed the expression of MMP-2 and avb3 integrin on the cell surface (Figure 1B). We further investigated the co-localization of MMP-2 and avb3 integrin during cell spreading. The fluorescence signal for MMP-2 was detectable on the cell surface 30 min after the cells were seeded onto the coverslip (Figure 1C upper panel). However, the signal of co-localization between MMP-2 and avb3 integrin was very weak during this time. At 3 h after seeding (Figure 1C lower panel), the cells partially spread out, and the fluorescence PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41591