Fig. 7. TNF--induced 3D scattering and invasion of collagen gels require metalloproteinase activity. (A,B) 2A4 cells were grown in collagen gels in defined medium for 3 days and subsequently incubated for an additional 6 days with 10 ng ml^-1 TNF- together with either the synthetic metalloproteinase inhibitor BB94 (1 µM) (B) or the inactive isomer BB1268 (3 µM) (A). Scale bars, 100 µm. (C) Quantification of the effect of increasing concentrations of BB94. 3D scattering is inhibited in a dose-dependent manner by BB94 (black columns) but is not affected by the inactive isomer BB1268 (open column). Quantification of 3D scattering was carried out after 6 days of treatment. Data represent the mean number of single cells per photographic field ± s.e.m. from at least three independent experiments. ^*P<0.01 compared with cultures incubated with TNF- alone. (D) Inhibition of collagen-gel invasion by BB94. 2A4 cells were plated onto a collagen gel and subsequently incubated with 10 ng ml^-1 TNF- in the presence of either BB94 or the inactive isomer BB1268. 5 days later, invasion was quantified by counting the number of all cellular structures (i.e. apparently single cells and cell cords) present in a photographic field at a focal plane beneath the surface monolayer. ^*P<0.05 compared with cultures incubated with TNF- alone; ^**P<0.0005 compared with cultures incubated with TNF- alone. (E) By gelatine zymography, proteolytic activity corresponding to the molecular weight of MMP-9 is not detectable in conditioned media from untreated 2A4 cells but is clearly present in conditioned media from TNF--treated cells. Conditioned media from murine J3B1A cells were used as a migration standard for MMP-9. The results illustrated were obtained with conditioned media collected from three independent experiments.