Fig. 1

Caspase-dependent programmed cell death of unattached oestrogen-responsive breast cancer cells. MCF-7, ZR-75 and EFM-19 cells were cultured in maintenance medium, trypsinised and added to uncoated or poly-HEMA-coated 35-mm-diameter wells in maintenance medium (10 % serum) or serum-free medium (Serum free) and cultured for 24 h (a). Cells were lysed after the indicated times and 10 μg protein aliquots were electrophoresed on 12 % polyacrylamide gels, transferred to nitrocellulose and the amount of 89 kDa cleaved PARP and GAPDH measured by western transfer analysis. Representative western transfer images are shown (b). The amount of each protein was determined by densitometric scanning of X-ray films. The amount of cleaved PARP was corrected for GAPDH expression with Labworks 4 software and is expressed as the percentage of the maximum value measured for each cell line (c). The mean values ± SEM are shown. Asterisks show times at which there is statistically significantly more cleaved PARP in the unattached cells than in attached cells (ANOVA, p < 0.01). EFM19 cells were incubated in serum-free medium in poly-HEMA-coated 22-mm-diameter wells, fixed, permeabilised and incubated with FITC-conjugated antibody against activated caspase 3 and analysed in a FACSCalibur as described in the Materials and Methods. Representative histograms are shown (d). Each experiment included triplicate samples and was replicated thrice