Figure 3
Induction of cell death in presence and absence of caspase-3 by PAC-1. (a) MCF7 (caspase-3-deficient) and MCFC3 (caspase-3-expressing) cells were stained with Hoechst 33 342 after PAC-1 (75 μ M) treatment with different time points. The representative images of Hoechst stained nuclei are shown and percentage of condensed nuclei are represented graphically (n=4, mean ±S.D.). MCF7 versus MCFC3, 24 h treatment (P<0.0015) and 48 h treatment (P<0.0003). (b) Clonogenic survival was assessed after PAC-1 treatment in both these cells and the representative images are shown. The clonogenicity was quantified by reading absorbance after staining with crystal violet as described. The graph represents clonogenic efficiency. (n=3, mean ±S.D.). PAC-1 treated MCF7 versus MCFC3 (P<0.0001). (c) MCF7 and MCFC3 cells were stained with TMRM after PAC-1 treatment and intensity was analyzed by Flow cytometry (20 000 events) to detect the Δψm loss in a time-dependent manner. Shifting of population to the left compared with that of the untreated population suggests loss in Δψm. (d) Caspase activation (caspase-3, -8, -2, -7, -9) and PARP cleavage were analyzed by western blot in both the cells after PAC-1 treatment with various time points
