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Supplementary Figure 1. Granzyme B cleaves caspase 3 to p20 but caspase processing is required for processing to p17.

Lysates from HeLa cells or HeLa-Bcl-2 cells treated with granzyme B (25 nM) and perforin (1 nM) for 2 h in the presence or absence of zVAD-fmk (100 mM) were subjected to Western blot analysis for caspase-3. Caspase-3 (p32), was cleaved to p20 and p17 fragments during granzyme B induced apoptosis. Cleavage of caspase-3 to the p20 subunit was caspase independent, however cleavage to the p17 subunit was blocked by zVAD-fmk

Supplementary Figure 2. Sustained loss of DYm is caspase dependent during apoptosis induced by native granzyme B or granzyme B delivered by pneumolysin O.

HeLa cells were treated with A) granzyme B (25 nM) isolated from YT cells and perforin (1 nM) or B) pneumolysin (2 ng/ml) for 4 h in the presence or absence of zVAD-fmk (100 mM). Cells were harvested, stained with TMRE and analysed by flow cytometry. PLO, pneumolysin.

Supplementary Figure 3. Time lapse microscopy of cytochrome c-GFP release and DYm during granzyme B induced apoptosis

HeLa cells expressing cytochrome c-GFP and stained with TMRE were treated with granzyme B (25 nM) and adenovirus (10 PFU) and followed by time-lapse confocal microscopy. The quicktime movie (SuppFigure3.mov) show the cells represented in Figure 4 a. In all cases, green represents cytochrome c-GFP and red indicates TMRE. The videos is compiled from 90 sequential frames taken 2 min apart and play at 8 frames per second.

Supplementary Figure 4. Time lapse microscopy of cytochrome c-GFP release and DYm during granzyme B-induced apoptosis in the presence of zVAD-fmk

HeLa cells expressing cytochrome c-GFP and stained with TMRE were treated with granzyme B (25 nM) and adenovirus (10 PFU) in the presence of zVAD-fmk (100 mM) and followed by time-lapse confocal microscopy. The quicktime movie (SuppFig4.mov) show the cells represented in Figure 4b. In all cases, green represents cytochrome c-GFP and red indicates TMRE. The videos is compiled from 90 sequential frames taken 2 min apart and play at 8 frames per second.