Fig. 2

Effect of loss of attachment on activation of signal transduction proteins in breast cancer cells. EFM-19 cells (a, b and c), MCF-7 (c) and ZR-75 cells (c) were trypsinised, added to 35-mm-diameter cell culture wells that had not (uncoated) or had been coated with poly-HEMA and cultured for the indicated lengths of time in serum-free medium. Cells were lysed and aliquots of 10 μg of protein were electrophoresed on 12 % polyacrylamide gels, transferred to nitrocellulose and the amounts of cleaved PARP, phosphorylated FAK, Akt, Bad, ERK1 and ERK2 and the corresponding total proteins and GAPDH were measured as described in the Materials and Methods. Representative images of the results obtained are shown (a and c) with the images for each phosphorylated protein above those of the equivalent total protein (c). The amounts of cleaved PARP and phosphorylated FAK were determined by densitometric scanning of X-ray films followed by analysis with Labworks 4 software and correction for GAPDH. The results obtained are expressed as a percentage of the maximum amount of cleaved PARP protein measured or phosphorylated FAK measured in attached (o) or unattached (•) cells. The bars show the standard errors of the mean. Asterisks show cleaved PARP that is statistically significantly more, or phosphorylated FAK that is statistically significantly less, in unattached cells than in attached cells (ANOVA, p < 0.01)