Fig. 5

Inhibition of the PI3K-Akt pathway restores TRAIL-induced apoptosis in FnIII-1c treated NCI-H460 cells. NCI-H460 cells were pre-incubated with the Akt inhibitor VIII (10 μM) or the PI3 kinase inhibitor LY294002 (10 μM) for 1 h. FnIII-1c (20 μM) was added for an additional hour and cells were stimulated with 100 ng/ml TRAIL for 2.5 h. PBS served as control. a Whole cell lysates were collected and subjected to western blot analysis for phosphorylated Akt (pS473Akt) and cleaved caspase 8. GAPDH was used as a loading control (b). Alternatively cells were evaluated for apoptosis by TUNEL assay and stained with Hoechst 33342 dye to visualize nuclei. Bar - 50 μm. c Quantification of the data shown in (b). TUNEL positive nuclei were determined in three independent experiments using Image J analysis. TUNEL positive nuclei were determined using Image J. Statistical analysis was done using a One-Way Anova with Tukey post-hoc analysis to determine significance. (*, p < 0.01; n.s = not significant)