Figure 2.(A) Similar chemical structure of AK3 and AK10. AK3 and AK10 both have a core piperazine group substituted with phenyl and benzoyl groups at the two piperazine nitrogens. (B) Increased caspase-3 and apoptosis in the combination treatment of AK3 or AK10 with TNF. HT29 cells were treated with AK3 or AK10 in the presence or absence of TNF. Cells were fixed, permeabilized and stained for active caspase-3. Higher proportion of cells stained for active caspase-3 in the combination treatment compared with control or AK3/AK10 treated cells. Positively staining cells displayed an apoptotic morphology. Bar, 100 μm. (C) Representative dose-response curves of caspase-3 activity with increasing concentrations of AK3 or AK10, in the presence or absence of TNF. Significant caspase activation was observed only in the combination treatment. The asterisks indicated points where the combination treatment was significantly higher than compound or TNF treatment alone (ANOVA, Tukey’s post-hoc test, p < 0.01). LD50s of AK3 and AK10 in the presence of TNF were determined to be 0.5 μM and 2.0 μM, respectively.