Figure 2

Induction of apoptosis by (A) GA and (B) di-GA in HL-60 cells. Cells were incubated with increasing concentrations of drugs for 24 h, and then double stained with Hoechst 33258 and propidium iodide. Afterwards cells were examined under the microscope with UV light connected to a DAPI filter. Nuclei with a morphological phenotype indicating apoptosis were counted and percentages of apoptotic cells were calculated. Experiments were conducted in triplicate. Error bars indicate s.e.m., asterisks significance (P<0.05). (C) Activation of caspase 3 and cleavage of PARP on treatment with di-GA. Logarithmically growing HL-60 cells were incubated with 10 μ M di-GA for 0.5, 2, 4, 8 and 24 h. Afterwards cells were lysed and protein expression was analysed by western blotting. The anti-caspase 3 antibody recognises only the cleaved peptide indicating its activation. Anti-PARP antibody recognises the full-length form (116 kDa) and the signature-type cleaved product (85 kDa) that is generated by active caspase 3. The antibody against β-actin was used to monitor equal sample loading.