Figure 2. Regulation of uPA production by TGF-ß1 and EGF in healthy and granulation-tissue fibroblasts. (A) Serum-free cultures of gingival fibroblasts from healthy and granulation-tissue (80,000 cells) were stimulated with increasing concentrations of TGF-ß1 or EGF for 48 hrs in DMEM without serum. Conditioned medium, normalized after the quantification of total protein amounts in the cell lysate at the end of the experiment, was analyzed through casein zymography. (B) uPA protein levels were determined through Western blot analysis of the concentrated conditioned medium of 500,000 cells stimulated with 10 ng/mL TGF-ß1 or 10 ng/mL EGF. (C) uPA-derived proteolytic activity was determined in conditioned medium of cells stimulated with 10 ng/mL TGF-ß1 or 10 ng/mL EGF through a radial diffusion assay. (D) PAI-1 production was identified through Western blotting of concentrated conditioned medium of cells stimulated with 10 ng/mL TGF-ß1 or 10 ng/mL EGF. (E) TGF-ß1-stimulated uPA production, determined through casein zymography, in each individual was normalized against non-stimulated uPA production (indicated as 1 in the graph) and compared among the 4 groups under study. Bars indicate averages ± standard deviations. Statistical analysis was performed with Student’s t test (n=33). (F) We performed Pearson correlation analysis, comparing TGF-ß1-stimulated uPA production through casein zymography and -SMA expression.