Figure 10.(A) Influence of AK3 and BI2536 on mitosis-dependent survivin phosphorylation. HT29 cells were treated with AK3 or BI2536 in the presence or absence of TNF as indicated. Lysates were analyzed by immunoblotting with antibodies against total survivin and survivin phosphorylated at Thr34. Actin was run as a loading control. (B) Effect of BI2536 on initiator caspase cleavage. HT29 cells were treated with TNF for the indicated times in the presence or absence of BI2536. Cell lysates were prepared and analyzed caspase-8 and caspase-9 cleavage. (C) Effect of BI2536 on gene activation by TNF. HT29 cells were treated with the TNF in the presence or absence of BI2536 as indicated. RNA was extracted at four hours and quantified for expression of TNF, BIRC3 and p21 mRNA by reverse transcription/real time PCR assay using actin as an internal control. The expression levels shown are relative to the untreated control cells. TNF activated the expression of the three genes tested relative to control cells (ANOVA, p < 0.001), but there was no significant effect of BI2536 on this activation. (D) Quantification of the live cells expressing TNFR1 on the surface. Images were captured and quantified for the number of cells staining with the TNFR1 antibody. Significantly more cells stained for surface expression of TNFR1 after BI2536 treatment (p < 0.01; Student’s t test).