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Fig. 2. The main gelatinolytic enzyme activity is localized to the stromal partner in cocultures of fibroblasts and CA1a cells and also in xenograft tumors, and contains significant levels of MMP-9 and MMP-2 activity. (A-C) Fibroblasts were labeled with DiI (red) before plating, fluorescein-labeled gelatin was used as a substrate (green). Nuclei were stained with DAPI (blue). The instrument settings of the microscope were kept constant for each experiment to allow comparison of the collected fluorescence signals of the different cultures. (A) In situ zymography of homotypic cultures and cocultures of DFs and CA1a cells. (B) Cocultures of MMP2KO or MMP9KO with CA1a cells showed significantly less enzyme activity than cocultures employing the corresponding wild-type fibroblasts. (C) Cocultures of human embryonic-lung fibroblasts (WI-38) and CA1a cells showed a growth pattern similar to the one in the heterologous cultures; however, presumably because of the slow growth of WI-38 cells, there were fewer fibroblasts. Gelatin zymography of culture supernatants of 4-day homotypic and heterotypic cultures showed an induction of MMP-9 in the coculture supernatant. (D) In situ zymography with DQ gelatin on serial sections of a xenograft tumor, formed by CA1A cells in nude mice, shows the main gelatinolyic activity (green) of the specimen localized in tissue that is also 
-smooth muscle actin-positive, thus exhibiting characteristics of stromal tissue. Tumor cells are cytokeratin-positive and show only a low gelatinolytic activity. Cytokeratin and -smooth muscle actin are detected with a secondary FITC-labeled antibody (green) by indirect immunofluorescence; nuclei are stained with DAPI (blue). Bars, 80 µm.
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