Figure 1.
Mapping the MCL-1ES domains crucial for mitochondrial apoptosis.
(A) The MCL-1ES mutants generated are shown. (B) A time course (6, 12, and 24 h) of cell viability of the MCL-1ES mutants in 293T cells is shown after transfecting equal amounts of the respective constructs. (C) Flow cytometry analysis of Annexin V-positive apoptotic cells was performed and (D) MMP of 293T cells was determined 24 h after transfection. Three independent experiments were performed, and the data are expressed as the mean ± SEM. Different letters denote statistically significant different values (P>0.05). (E) The activation of caspases 9, 8, and 3 was assessed by immunoblot analysis using transfected 293T cells. (F) Caspase 3 activity was measured using the DEVD peptide conjugated to p-nitroaniline as described in the Material & Methods. The values are expressed as the mean ± SEM of three replicates. (G) The cytosolic release of cytochrome c was assessed in transfected 293T cells. The cells were separated into mitochondrial and cytosolic fractions. Adequate fractionation was demonstrated by immunoblot analysis using anti-cox IV and anti-β-actin antibodies. The relative cytochrome c level (left graph) was quantified, and MCL-1ES localization (right graph) in the mitochondrial and cytoplasmic fractions was presented (lower panel). The values are expressed as the mean ± SEM of three independent experiments.
Figure 2.
The BH3 domain of MCL-1ES is critical for its apoptotic activity.
(A) A time course of cell viability of MCL-1ES and the BH3 mutant (BH3M), (B) Annexin V-positive apoptotic cell analysis, (C) western blot analysis of caspase activation, and (D) caspase 3 activity analysis were performed in 293T cells transfected with WT or the BH3M MCL-1ES mutant as described in the Materials and Methods. The values are expressed as the mean ± SEM of three independent experiments. Different letters denote statistically significant different values (P>0.05). (E) Changes in cytochrome c release and intracellular localization of BH3M were determined by subcellular fractionation followed by western blot analysis (upper panel). The levels of cytochrome c (left graph) and the MCL-1ES protein (right graph) in the mitochondria and the cytoplasm were quantified and are presented (lower panel). (F) Confocal microscopic images of cells overexpressing WT (upper panel) and BH3M MCL-1ES (lower panel) are shown. The cells were stained with an anti-Flag antibody and MitoTracker.
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.
Figure 4.
The importance of MCL-1L in MCL-1ES-mediated apoptosis.
(A) The interactions between MCL-1L and WT or BH3M MCL-1ES were analyzed by immunoprecipitation in 293T cells followed by western blot analysis. Equal amounts of total protein from cell lysates were used in each lane. (B) A time course of cell viability, (C) Annexin V-positive apoptotic cells, and (D) caspase 3 activity were analyzed in 293T cells that over- or under-expressed MCL-1L after transfecting with WT or BH3M MCL-1ES. Equal amounts of plasmid DNA were used. Data are from two independent experiments performed in triplicate. The percentage of viable 293T cells is expressed as the mean ± SEM. Different letters denote statistically significant different values (P>0.05). (E) Changes in MCL-1ES oligomerization were assessed in 293T cells that over- or under-expressed MCL-1L. (F) The influence of MCL-1L on intracellular MCL-1ES localization was determined by immunofluorescent confocal microscopy. 293T cells were stained with anti-MCL-1L and anti-Flag (MCL-1ES) antibodies and visualized with goat anti-mouse IgG or goat anti-rabbit IgG. (G) The influence of MCL-1L on cytochrome c release by MCL-1ES and BH3M and the intracellular localization of MCL-1ES and BH3M were determined by subcellular fractionation and western blot analyses (left panel). The quantified results shown in the graphs were from three independent experiments.