Figure 6.

p53-induced apoptosis in the Vm10 cell line is inhibited by TSA. Caspase-3 activity was analyzed in the Vm10 cell extracts indicated; cells were cultured at 39°C (mutant p53) or 32°C [wild-type (wt) p53, apoptosis] in the presence or absence of 100 nm TSA for 24 hr. As positive controls, Vm10 cells were cultured at 39°C in the presence of 100 nm staurosporine (Calbiochem), TNFα (10 ng/ml, RD Systems) plus cycloheximide (CHX, 40 μg/ml), or dilution vehicle alone (DMSO, 39°C). The activity of caspase-3 is measured as the ability of cell extract to catalyze the cleavage of AMC–DEVD and release the AMC fluorochrome. Readings were performed in duplicate; in the case of temperature-shifted cells, the data plotted are the average results of three independent experiments, with standard deviations plotted on the error bars. For staurosporine and TNFα-treated cells, the data plotted are the averages of two independent experiments. As a negative control, an inhibitor specific for caspase-3 (Ac–DEVD–CHO) was also added to the cell extract (light stippled bars).