Figure 5.

Bivalent IAP antagonists inhibited the nuclear translocation and the transcriptional activity of p65/NF-κB following TNF stimulation. (a) The nuclear translocation of p65/NF-κB in HeLa cells was evaluated using the Operetta High Content Imaging System (PerkinElmer, Waltham, MA, USA), as described in Materials and Methods section. The nuclear DNA was counter-stained with Hoechst 33342: the blue nuclear stain is shown in each of the bottom panels. Arrows indicate p65/NF-κB localized in cytoplasm (perinuclear) or nuclei in unstimulated (left panel) and TNF-stimulated (right panel) HeLa cells, respectively. Note the fluorescence intensity of p65/NF-κB increased in some of the cells stimulated with TNF, whereas the perinuclear p65/NF-κB intensity was reduced following TNF stimulation. (b) Bivalent IAP antagonist showed efficient inhibition of the nuclear translocation of p65/NF-κB in HeLa cells. Quantitative evaluation of p65/NF-κB in the nuclear or cytoplasm in HeLa cells using Operetta High Content Imaging System. Cells were treated with IAP antagonist for 2 h, followed by TNF stimulation. Representative result from three independent experiments (mean±S.E.). (c) Bivalent IAP antagonist showed efficient inhibition of the transcriptional activity in the p65/NF-κB reporter gene assay. The transcriptional activity of p65/NF-κB was evaluated using HeLa cells stably harboring p65/NF-κB reporter gene. Results are mean±S.E. (d) Despite efficient degradation of GFP-cIAP1, monovalent IAP antagonists and B3-EL4 were less effective at inhibiting the activation of p65/NF-κB following TNF stimulation in comparison with bivalent IAP antagonists.