Figure 7. Effect of AK3 on gene activation and initiator caspase cleavage by TNF. (A) HT29 cells were treated with AK3, TNF or AK3 plus TNF for 4 h. RNA was isolated, reverse transcribed and quantified for expression of TNF, BIRC3 and p21 by real-time PCR. TNF activated the expression of the three genes tested relative to control cells (ANOVA, p < 0.001), and there was no significant effect of AK3 on this activation. (B) AK3 enhances the TNF induced cleavage of caspase-8 and caspase-9. Cells were treated with TNF for 0, 2, 4 and 6 h in the presence of absence of AK3 and lysates were analyzed by immunoblotting with antibodies against full length and cleaved caspases-8 and 9. (C) Protein samples were analyzed for TNFR1 by immunoblotting. β-Actin was used as the loading control.