FIGURE 6.

A, enhanced adhesion on vitronectin of EGF- and lactoferrin-stimulated HT1080 cells. A, serum-starved HT1080 cells were incubated in the absence (vehicle) and presence of PD98059 (25 μm) for 30 min and subsequently stimulated with EGF (25 ng/ml) and/or lactoferrin (70 μg/ml) and plated on vitronectin-coated plastic dishes. After 10 min, the non-adherent cells were removed by aspiration, and the adherent cells were stained with crystal violet. Cell adhesion was quantified by determining the absorbance at 540 nm in an ELISA plate reader. Values represent mean absorbance ± S.E. (error bars) (n = 4). Statistical significance was assessed by analysis of variance followed by Dunnett's multiple comparison test, which documented a statistically significant effect of EGF + lactoferrin versus control and a statistically significant inhibition of this stimulation by PD98059 (p < 0.01). B, long term stimulation with EGF and lactoferrin enhances migration of HT1080 cells on vitronectin. HT1080 cells were seeded on vitronectin-coated glass coverslips and were stimulated with either EGF (25 ng/ml) or lactoferrin (LF; 70 μg/ml) alone or in combination. The coverslips were mounted onto cell culture chambers on a heated stage insert on an Olympus AX-70 microscope for recordings by time lapse microscopy of cell migration for over 15 h with 10 images recorded per hour (F-View digital camera, Soft Imaging System). Recorded time lapse sequences were analyzed by assessing the length of the track of individual cells. Data are based on the analysis of the trajectories of 90 cells recorded in three independent experiments. The effect of lactoferrin + EGF was statistically significant in relation to all other values (p < 0.05, analysis of variance followed by Dunnett's test for multiple comparisons).