Figure 3.

Bid^−/− and Bax × Bak^−/− MEFs do not release cyt c but are susceptible to GzmB-induced death. (A) Wt and Bid^−/− MEFs were harvested from d13.5 embryos and used as target cells in the reconstituted death assay, as described (4), or were pretreated with 50 μM zVAD-fmk for 30 min before treatment with rGzmB plus LAK extract. Cells were treated in duplicate with increasing concentrations of rGzmB plus LAK extract; viability was defined as described (4). (B) Wt and Bax × Bak^−/− (DKO) MEFs were generated as described (19). Both wt and DKO cells, with or without pretreatment with 50 μM zVAD-fmk, were used as targets in the reconstituted death assay (4) as described in A. All experiments in A and B were performed at least three times with similar results. (C) Cyt c distribution in wt (Bid^+/+) and Bid^−/− MEFs. Wt (Left) or Bid^−/− MEFs (Right) were untreated (control) or treated with rGzmB plus LAK extract (GzmB) for 2 h. Immunofluorescence for cyt c was performed as described (34) and is displayed in the left of each set. 4′-6-Diamidino-2-phenylindole staining for nuclear morphology of the same cells is shown in the right of each set. (D) Cyt c distribution in wt and DKO MEFs. Single planes from deconvoluted z axis stacks of cyt c immunofluorescence in wt and DKO MEFs untreated (labeled control) or treated with LAK extract and rGzmB (labeled GzmB). The cyt c subcellular distribution patterns shown are representative of >50% of the cells analyzed. (Bar = 7 μm.)