MT3-MMP in Matrix-Dependent Melanoma Invasion Figure 4. MT3-MMP induces fibrin invasion but inhibits collagen invasion of Bowes melanoma cells. (A) The stable cell pools transfected with MT3-MMP cDNA (MT3-MMP) or mock vector (Mock) were allowed to invade 3D fibrin or collagen gels for 5 d. Quantitative results are expressed as the number of invasive cells per microscopic field relative to the mock-transfected cells (n = 3, *p = 0.02). White dotted lines in the representative light micrographs of fibrin and collagen cross-sections mark the level below which the invaded cells were counted. (B) Chart shows average MT1- MMP and MT3-MMP mRNA expression relative to mock-transfected cells as assessed by qPCR. (C) The stable mock or MT3-MMP expressing cells were treated with GM6001 (10 mM) and bafilomycin A (100 nM) for 16 h as indicated, followed by MT3-MMP and MT1-MMP detection by immunoblotting. Full-length and processed forms of MT3-MMP were detected as 65/60 kDa and 37 kDa bands, respectively, and MT1-MMP as 60 kDa and 37–43 kDa bands. Tubulin served as a loading control. Asterix marks a non-specific band. (D) Cell surface MT3-MMP was detected by surface biotinylation of the stable cells on fibrin and collagen or cells transfected with cDNA for MT3-MMP or catalytically inactive MT3-MMP (MT3E/A) followed by MT3-MMP immunoprecipitation and detection with peroxidase-conjugated streptavidin. Cells were treated with GM6001 (10 mM) for 16 h prior to surface biotinylation where indicated. Asterix marks a non-specific band. (E–F) Bowes cells expressing the indicated cDNAs were lysed, followed by immunoblotting or immunoprecipitation. GM6001 (GM, 10 mM) was added where indicated. Ponceau staining and tubulin served as a loading control. Asterix marks non-specific bands. doi:10.1371/journal.pone.0028325.g004 MT3-MMP, or both proteases in combination. Consistent with the fact that both MT1-MMP and MT3-MMP can degrade fibrin [18], either MT1-MMP or MT3-MMP conferred the cells with the ability to invade fibrin gels and displayed additive effects when co-expressed (Fig. 5A). In contrast, while MT1-MMP alone induced COS-1 cell invasion into collagen matrices, MT3-MMP, PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28325