MMP-2 Promotes avb3 Mediated Tumor Cell Migration Figure 1. Expression of MMP-2 and avb3 in human A375 melanoma cells. (A) Cells were suspended in PBS and labeled with anti-avb3 (red) and anti-MMP-2 (green) antibodies. Scale bar = 10 mm. (B) Flow cytometric analysis of avb3 integrin and MMP-2 expression on the cell surface. The expression of avb3 integrin and MMP-2 was evaluated using anti-avb3 and anti-MMP-2 antibodies, and isotype IgG was used as a negative control. (C) A375 cells were seeded onto coverslips coated with human fibronectin for 30 min (upper panel) and 3 h (lower panel), then labeled with anti- avb3 (red) and anti-MMP-2 (green) antibodies. Scale bar = 10 mm. (D) A375 cells in agarose drops were seeded onto coverslips coated with human fibronectin. The cells were labeled with anti-avb3 (green) and anti-MMP-2 (red) antibodies 24 h after seeding and observed under a confocal microscope. Scale bar = 10 mm. (E) A375 cells were fluorescently stained and seeded on 96 well flat-bottom plates coated with APMA-activated recombinant human MMP-2 or 0.5% BSA. The control cells were treated using RGD peptides. doi:10.1371/journal.pone.0041591.g001 signals of MMP-2 and avb3 integrin appeared stronger on the cell surface and they were partly co-localized. An agarose drop model was used to detect the co-localization of MMP-2 and avb3 integrin in the migrating cells (Figure 1D). After 24 h, both MMP-2 and avb3 integrin were strongly expressed on the surface of cells and partially co-localized with each other. A static adhesion assay was employed to detect the association between MMP-2 and avb3 integrin on A375 cells. As shown in Figure1 E, approximately more than 59% tumor cells adhered to the culture wells coated with APMA-activated MMP-2 compared with those cells blocked with RGD peptides. These results demonstrated that MMP-2 and avb3 integrin were expressed on A375 cells, and they might interact with each other. PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41591