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. 1997 Jul 22;94(15):7959–7964. doi: 10.1073/pnas.94.15.7959

Figure 2.

Figure 2

Activation of MMP-2 and degradation/invasion of fibronectin-coated crosslinked gelatin substrata by melanoma cells treated with Con A or overexpressing MT-MMP. (A) Activity of MMP-2 released into the medium (Medium) or associated with cells (Cell lysate) in the presence (Con A) or absence (Parent) of Con A or transfected with pSG5-MT-MMP (MT-MMP) were analyzed by gelatin zymography. The three bands shown represent 72-kDa pro-MMP-2, the 62-kDa intermediate form, and the 59-kDa active form of MMP-2. (B) Activity of MMP-2 released into the medium (Conditioned medium) by the cells or associated with cells transfected with pSG5-MT-MMP (Cell lysate) in the absence (MT-MMP) or presence of the following protease inhibitors: 100 nM CT1847 (InMMP), 200 nM CT1847 (InMMP′), 320 μg/ml ɛ-amino caproic acid (ACA), 5 μg/ml pepstatin A (PSA), and 10 μg/ml leupeptin (LP). Gelatinase activities were analyzed by gelatin zymography. (C) Fibronectin degradation by cells transfected with pSG5 plasmid (Mock), treated with Con A, or transfected with pSG5-MT-MMP (MT-MMP). Cells were allowed to spread on fluorescein-fibronectin-coated crosslinked gelatin films and were photographed with fluorescence microscopy after 1, 3, 6, and 12 h. Wide black areas in the lower panels indicate the degradation of fibronectin-gelatin films by cells. Mock transfectants and the cells treated with Con A showed very little black area on the film, indicating background levels of fibronectin degradation by the melanoma cells, whereas cells transfected with MT-MMP show much higher levels of degradation. Bar = 25 μm. (D) The areas of degraded fibronectin substratum under the cells transfected with pSG5 plasmid (Mock) or transfected with pSG5-MT-MMP (MT-MMP) in the absence or presence of Con A were measured with Optimas image analyzer after 6 h. Each value represents the mean of three separate determinations (among 100 cells) ± SD. Duplicate experiments gave similar results. (E) The invasiveness of the cells transfected with pSG5 plasmid (Mock) or transfected with pSG5-MT-MMP (MT-MMP) in the absence or presence of Con A after 12 h was analyzed in terms of percentage of cells forming invadopodial extensions in the crosslinked gelatin film. Each value represents the mean of three separate determinations (among 100 cells) ± SD. (F) The areas of degraded fibronectin substratum under the cells in the presence of protease inhibitors as indicated above were measured with Optimas image analyzer. Each value represents the mean of three separate determinations (among 100 cells) ± SD.