Fig. 3

FnIII-1c stimulates the PI3K-Akt-dependent activation of the αvβ5 integrin. a NCI-H460 cells were seeded in complete medium for 48 h and then immunostained (FITC) for the indicated integrins. Positive staining was seen only for αvβ5 integrin. Panel A' shows αvβ5 integrins in focal contacts of individual cells. Bar = 10 μm. Nuclei were counterstained with Hoechst 33342. b NCI-H460 cells were pretreated with 10 μM FnIII-1c or PBS for 1 h before seeding onto wells coated with the designated concentrations of vitronectin. After 1 h, cell adhesion was measured by toluidine staining. The data are presented as the mean absorbance (OD) ± SE of at least 3 independent experiments. Results were analyzed by a two-way Anova with Tukey’s post-hoc analysis (*, p < 0.05). c H460 cell monolayers were incubated with blocking antibody to the αvβ3 (LM609), αvβ5 (P1F6) integrin or control mouse IgG. Cells were then treated with 10 μM FnIII-1c for 1 h prior to stimulation with 100 ng/ml TRAIL for an additional 2.5 h. Lysates were immunoblotted for cleaved caspase 8 and GAPDH. Cells treated with PBS or FnIII-1c served as additional controls. A representative blot is shown. d Densitometric quantification of the data shown in (c). Data are presented as the amount of cleaved caspase 8 relative to TRAIL-treated cells, which was set at 1. Each bar represents the mean ± SE of three independent experiments. A one-way ANOVA with Tukey post-hoc analysis was used to determine statistical significance (*, p < 0.05). e NCI-H460 cells were pre-treated with 10 μM VIII (Akt1/2 kinase inhibitor) or 10 μM LY294002 (PI3K inhibitor) for 30 min before treatment with FnIII-1c. Cells treated with PBS served as control. Treated cells were seeded onto plates coated with vitronectin (0.5 μg/ml) and allowed to adhere for 1 h. Adhesion was measured by toluidine staining (OD) and presented as fold-change relative to FnIII-1c-treated cells, which was set at 1. The data represent the mean ± SE of three independent experiments. Results were analyzed by a two-way Anova with Sidak’s multiple comparison tests (*, p < 0.05)