FIGURE 2.

Exposure of the cryptic antiadhesive site in fibronectin is responsible for serum starvation-induced anoikis. NIH3T3 cells were cultured under various conditions in serum-free medium for 1 or 2 days, and cell viability was determined as described under “Materials and Methods.” A, NIH3T3 cell suspension with or without 9EG7 (20 μg/ml) were seeded onto a 96-well plate coated with either fibronectin alone (0.25 μg/ml) (a) or fibronectin (0.25 μg/ml) and FNIII14-OVA (0, 50, or 200 μg/ml) (b). Cells were cultured with serum-free medium in the presence (a) or absence (b) of peptide FNIII14 at the indicated concentrations (0–100 μg/ml). *, p < 0.01 compared with day 0. **, p < 0.05 compared with the value without FNIII14 on day 2; ***, p < 0.01 compared with control. B, cells were seeded onto a 96-well plate coated with fibronectin and then cultured with or without the following factors: IgG, preimmune normal IgG (20 μg/ml); α-HepII, anti-heparin-binding domain II of fibronectin (FNH3–8) (20 μg/ml); α-FNIII14, anti-FNIII14 function-blocking antibody (0, 10, or 20 μg/ml). *, p < 0.01 compared with day 0; **, p < 0.05 compared with the value without antibody on day 2. C, cells were seeded onto a 96-well plate coated with fibronectin and then cultured with or without the following factors: BB-94, a broad spectrum MMP inhibitor (50 or 150 nm); BiPS, (2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide, a specific inhibitor of MMP-2 and MMP-9 (2.5 or 10 μm). *, p < 0.01 compared with day 0; **, p < 0.05 compared with the value without MMP inhibitors on day 2. Each point represents the mean ± S.D. of triplicate determinations. One of three or four individual experiments is shown. D, gelatin zymography of culture supernatant of NIH3T3 cells. NIH3T3 cells (1 × 10⁴ cells in lane 1 and 5 × 10⁴ cells in lane 2) were cultured on the fibronectin substrate with serum-free medium for 18 h. An aliquot of the supernatant was subjected to gelatin zymography. Data shown are representative of two individual experiments.