Fig. 6

Effect of inhibition of the PI3-kinase/Akt pathway on the protection of breast cancer cells from anoikis by IGF-1. Cells were trypsinised, resuspended in serum-free medium alone or with 50 ng/ml IGF-1 (A and B) or 10 ng/ml IGF-1 (c and d) and in the absence or presence of 20 μM LY294002 (a and b) or 100 nM GSK690693 (c and d) and then placed in 35-mm-diameter cell culture wells that had been coated with poly-HEMA and cultured for 24 h. Cells were lysed and the amount of phosphorylated IGF receptors, phosphorylated Akt, phosphorylated GSK3β, type I IGF receptor, Akt, GSK3β, cleaved PARP and GAPDH were measured by western transfer in aliquots of 10 μg of protein. Representative images are shown with the images for each phosphorylated protein above those of the equivalent total protein (a and c). The means ± SEM from three experiments are shown. Statistical significance of differences between untreated cells and cells treated with IGF-1 (*), or between cells treated with IGF-1 alone and in the presence of the inhibitor (**) are indicated (p < 0.05; ANOVA)