Resistance to caspase-independent cell death requires persistence of intact mitochondria.

Cell treatments

To induce apoptosis, cells were treated with the indicated concentrations of staurosporine (Sigma), human TNF (Peprotech), actinomycin D (Sigma) or UV irradiated using a Stratagene UV crosslinker. Where indicated, the pan-caspase inhibitor Q-VD-OPh was added to a final concentration of 20μM (EMD Biosciences). ABT-737 and enantiomer were obtained from Abbot Laboratories. Where indicated, cells were incubated with 25μM FCCP (Sigma) for 1 hour prior to washing and re-addition of culture medium.

Microscopy

Cells were plated on dishes containing fibronectin coated coverslips (Mattek) 24 hr prior to treatment and imaged using a spinning disk confocal microscope (Zeiss). For time-lapse experiments, media on the cells was supplemented with HEPES (20 mM) and 2-mercaptoethanol (55 μM) prior to imaging.

Clonogenic survival assays

Following overnight treatment, HeLa cells expressing Smac GFP and GAPDH were sorted into the lowest and highest 20% fluorescent populations 2×10 5 cells from both populations were washed and plated in a 12-well plate. For the ABT-737 clonogenic assays, 2.5×10 3 cells/per well were plated down on a 12-well plate two days prior to treatment. The following day, cells were treated with ABT-737 or enantiomer overnight (both at 1μM) then treated with ABT-737 or enantiomer plus STS +/− Q-VD-OPh for 8 hours. Where noted, ABT-737 or enantiomer was added at 1μM for 16 hours post-treatment. The following, day cells were washed and incubated with new media +/− Q-VD-OPh. Where added, Q-VD-OPh was refreshed for the next two days. Following clonogenic outgrowth, colonies were visualized by staining with methylene blue 0.1% w/v in methanol/water 50% v/v for 20 minutes.

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