Figure 1.

ABT-737 restores cell death of HeLa-Bcl-2 cells treated with human NK cells or GraB. HeLa-Bcl-2 cells were treated with (a) human NK cells (stimulated with 25U IL-2 for 4 days), or (b) Pfp (1 nM) and GraB (25 nM) in the presence of zVAD-fmk (100 μM). ABT-737 (500 nM) was added at t=0 and cells were fixed after 4 h. Images of morphology (DIC) and cyt c location (immunofluorescence) were taken using an Olympus CellR fluorescence microscope with a × 40 oil-immersion lens. Arrows indicate cells that have released cyt c and asterix are cells that have not. (c) HeLa-Bcl-2 cells were treated with Pfp (1 nM)/GraB (25 nM) in the presence or absence of ABT-737 (500 nM) or Enantiomer (500 nM). Cell death was determined by Annexin V binding. Data are the average±S.E.M. for three independent experiments. (d) HeLa-Bcl-2 cells were treated with Pfp (1 nM)/GraB (25 nM) in the presence or absence of ABT-737 (500 nM) ± GraB inhibitor (C20; 10 μM) for 4 h. Cell death was determined by release of ⁵¹Cr that had been pre-loaded into the target cells. Data are the average±S.E.M. for three independent experiments. (e) HeLa-Bcl-2 cells were pre-labelled with ⁵¹Cr and incubated with Pfp (1 nM)/GraB (12.5 nM) together with the concentration of ABT-737 indicated. Media containing 10% FCS was added at 1 h. Specific ⁵¹Cr released from the target cells was measured at 4 h as an indication of cell death. Data are the average±S.E.M. of five experiments. (f) ⁵¹Cr release from HeLa-Bcl-2 cells treated with Pfp (1 nM)/GraB (12.5 nM) for 4 h. ABT-737 (500 nM) was either omitted (No. ABT-737) or added to the HeLa-Bcl-2 cells as indicated either at 0 min (ABT-737, t=0) or after 60 min (ABT-737, t=60)