Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - TRAIL induces a time-dependent processing of caspase-8. MCF-7 cells were exposed to TRAIL (1 μg ml^-1) and the processing of caspase-8 assessed by western blotting using an antibody that detects the unprocessed zymogen and the processed catalytically active large subunit. The p18 large subunit was detected after 15 min of treatment.
• Supplemental Figure 2 - Cleaved caspase-8 can be translocated into the nucleus. MCF-7 cells were permeablized with digitonin (40 μg ml^-1) for 5 min on ice and then incubated with recombinant active caspase-8 (600 ng ml^-1) for a further 30 min at 37°C. Where indicated, nuclei were pre-treated with WGA (50 μg ml^-1) for 15 min at room temperature prior to incubation with caspase-8. The cells were then fixed and labelled with either the cleaved caspase-8 antibody (green), which labels only the processed p10 subunit of caspase-8 or with a second caspase-8 antibody that detects the intact zymogen, the p43/41 and p18 processed forms. The nuclei were also stained with Hoechst 33258 (blue). Bar, 15 μm.These results show that cleaved caspase-8 can enter the nucleus and this can be prevented by pre-treatment with WGA.
• Supplemental Figure 3 - Cleaved caspase-8 does not co-localize with PIKA or PML bodies. (A) MCF-7 cells were treated with TRAIL (1 μg ml-1) for 1 h and labelled with antibodies against PIKA (red), cleaved caspase-8 (green) and Hoechst (blue). Arrow indicates the PIKA body and shows that there was no co-localization of the signals. (B) A549 cells were treated with TRAIL (1 μg ml-1) for 1 h and labelled with antibodies against PML protein to highlight the PML bodies (red), cleaved caspase-8 (green) and Hoechst 33258 (blue). Bar, 8 μm.
• Supplemental Figure 4 - Staurosporine induces a time dependent loss of CENP-C labelling. MCF-7 cells were exposed to staurosporine (STS 1 μM) for 4-6 h either alone or in the presence of z-VAD.fmk (50 μM). Cells were then labelled with an antibody against CENP-C (554) (red) and stained with Hoechst 33258 (blue). Staurosporine induced a time-dependent loss of CENP-C labelling that was prevented by z-VAD.fmk, indicating that caspase activation rather than protein kinase inhibition was responsible for the loss of CENP-C labelling.
• Supplemental Figure 5 - Knockdown of caspase-7 protects against the loss of CENP-C. (A) MCF-7 cells were transfected for 24 h with either scrambled, XIAP or three different caspase-7 siRNA oligonucleotides (100 nM) and then re-transfected 24 h later. All three caspase-7 oligonucleotides caused depletion of caspase-7 but did not affect levels of caspase-8 and –6. (B) Bcl-xL over-expressing MCF-7 cells, used to minimize involvement of the intrinsic pathway, were exposed to TRAIL (1 μg ml^-1) for 3 h following transfection of the cells as described in (A) but using only oligonucleotide 3 caspase-7 siRNA (100 nM). Following labelling of the cells with CENP-C antibodies, confocal microscopy was used to score the number of cells without label. There was a highly significant decrease (p<0.01) in the median % of cells without CENP-C following exposure to TRAIL for 3 h (n=3).