Figure 3

Staurosporine induced apoptosis activation of the caspase cascade in both MCF-7 and T47D cells. (A) Cells were pre-incubated with 100 μM z-VAD-fmk (Z-VAD) for 1 h prior to the addition of 1 μM STS for 16 h. Cells were fixed, permeabilised and DNA fragmentation detected by ISNT. Error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated for different time periods with 1 μM STS. Cell lysates were prepared and incubated with Ac-DEVD-pNA (caspase-3/7). The reaction products were measured at 405 nm after a 4 h incubation. Error bars represent the mean±s.e.m. of four independent experiments where * indicates statistically significant increase in DEVDase activity compared to untreated control (0 h), (ANOVA: F=3.38, P<0.05, d,f 3,12. Tukey multipe comparison test, P<0.05). (C) Full length caspase-6 protein is cleaved in MCF-7 but not in T47D cells after treatment. Cells were treated with 1 μM STS for indicated times, lysates prepared and 50 μg protein subjected to SDS–PAGE followed by Western transfer and probed with caspase-6 antibody. C indicates untreated cells. The results are representative of three independent experiments.