Figure 3. BAK- and BAX-independent apoptosis induced by MCL-1ES via its mitochondrial oligomerization.

(A) A time course of cell viability was performed in wild-type (WT), bak^−/−, bax^−/−, and bax^−/−bak^−/− knockout MEF cells after transfection with WT or BH3 mutant (BH3M) MCL-1ES. (B) A flow cytometry analysis of Annexin V-positive apoptotic cells was performed, and (C) MMP, (D) cytochrome c release to cytosol, (E) caspase-3 activity, and (F) formation of apoptosome complexes were determined in bax^−/−bak^−/− MEF cells transfected with WT or BH3M MCL-1ES as described in the Materials and Methods. The values (mean ± SEM) were determined from three independent experiments. (G) The lack of MCL-1ES-induced BAK or BAX oligomerization was verified in 293T cells. (H) Oligomer formation of MCL-1ES in 293T cells and (I) in bax^−/−bak^−/− MEF cells was assessed. The heavy membrane fraction was obtained and cross-linked with glutaraldehyde. Oligomer formation was determined by western blot analysis using the appropriate antibodies.