BAX/BAK Independent Cell Death Figure 2. BH3 peptides induce cell death in the absence of BAX, BAK and BOK. (A) Western blot demonstrating levels of BOK protein levels in Bax2/2/Bak2/2 MEFs stably transfected with shRNA against BOK. Stable Bax2/2/Bak2/2 MEFs expressing a shRNA hairpin against D. melanogaster HIF (dHif) served as a control. (B) Bax2/2/Bak2/2 MEFs stably transfected with a shRNA against BOK or dHIF were treated with BID BH3 peptide (100 mM), mutant BID BH3 peptide (100 mM) or BIM BH3 peptide (80 mM) and after 1 hour the percentage of PI positive cells was analyzed by FACs analysis. Mean values6SEMs of 3 independent experiments are shown. doi:10.1371/journal.pone.0005646.g002 BH3 peptides induce a decrease in mitochondrial membrane potential in Bax2/2/Bak2/2 MEFs independent of the permeability transition pore Typically, cells undergo a loss of mitochondrial membrane potential upon cytochrome c release [26]. To assess mitochondrial membrane potential in wild-type or Bax2/2/Bak2/2 MEFs, we loaded cells with fluorescent dye tetramethylrhodamine ester (TMRE). BIM and BID BH3 peptide induced a decrease in TMRE fluorescence compared to the mutant BID BH3 peptide in both wild-type or Bax2/2/Bak2/2 MEFs indicative of mitochon- drial membrane depolarization (Figure 6A and B). To test whether the permeability transition pore (PTP) is responsible for the loss in mitochondrial membrane potential, we utilized the release of calcein from the mitochondria. Bax2/2/ Bak2/2 MEFs were loaded with calcein and cobalt chloride, which quenches calcein signal in the cytosol. Thus the remaining signal is calcein retained in the mitochondria. Calcein is released upon PTP opening with agents such as ionomycin. BID BH3 peptide treated Bax2/2/Bak2/2 MEFs had comparable mitochondrial calcein retention to that of DMSO and mutant BID treated Bax2/2/ Bak2/2 MEFs, which could be decreased to baseline upon the addition of ionomycin (Figure 6C). We further assessed for the involvement of the PTP in the peptide induced cell death by its direct inhibition using 1 mM of cyclosporine A (CsA). Inhibiting the PTP with CsA did not protect Bax2/2/Bak2/2 MEFs from peptide induced cell death (Figure 6D). We confirmed that 1 mM CsA was efficient at inhibiting the permeability transition pore by assessing for calcein retention in the mitochondria following treatment with ionomycin (Figure S3). Calcium is one of the major regulators of PTP opening. We tested whether calcium was involved in the BH3 peptide induced cell death in the Bax2/2/Bak2/2 MEFs. The calcium chelator BAPTA-AM did not prevent BID or BIM BH3 peptide induced cell death (Figure 6E). These data suggest that BH3 peptides induced mitochondrial membrane depolarization without invoking PTP opening. BH3 peptides induce mitochondrial fragmentation in Bax2/2/Bak2/2 MEFs We observed a change in mitochondrial morphology from long tubular to a small punctuated pattern of cytochrome c-GFP in Bax2/2/Bak2/2 MEFs (Figure 4C). To further confirm these morphological changes in mitochondria, Bax2/2/Bak2/2 MEFs were stained with a fluorescent dye, Mitotracker CMX-ROS. Bax2/2/Bak2/2 MEFs treated with mutant BID BH3 peptide displayed normal mitochondrial dynamics, and mitochondria appeared tubular and elongated (Figure 7A top panel). By contrast, Bax2/2/Bak2/2 MEFs treated with the BID and BIM BH3 peptides displayed short, spherical mitochondria (Figure 7A middle and bottom panel respectively), indicative of mitochondrial fragmentation. Bax2/2/Bak2/2 MEFs treated with BAD or NOXA BH3 peptides, did not reveal any changes in the mitochondrial morphology and maintained the elongated tubular structures (Figure 7B). However, the combination of BAD and NOXA peptides induced a change in the mitochondrial morphology from tubular and elongated structures to smaller spherical structures (Figure 7B) similar to those observed with BID and BIM. As a control, the combination of NOXA with a mutant BAD peptide did not induce changes in mitochondrial structure (Figure 7B, bottom panel). These data demonstrate that mitochondria underwent fragmentation only when the pro- survival proteins were neutralized with BH3 peptides. To further confirm that the change in mitochondrial morphol- ogy was not dependent on peptide uptake by the cell, we performed microinjection of BID BH3 and mutant BID BH3 peptides that did not contain the 8-D arginine sequence in combination with FITC dextran as a marker for injected cells. We captured images of cells that were injected next to non injected cells to show the difference in mitochondrial morphology. As shown in figure 8A when Bax2/2/Bak2/2 MEF were microin- jected with the BID BH3 peptide, the mitochondrial morphology changed from long tubular structures to small spherical structures, while the mutant BID BH3 peptide did not induce a change in mitochondrial structure (Figure 8B) upon microinjection. BH3 peptides induce a decrease in mitochondrial aspect ratio of Bax2/2/Bak2/2 MEFs We further verified the change in mitochondrial morphology using electron microscopy. Electron micrographs revealed that Bax2/2/Bak2/2 MEFs treated with the mutant BID BH3 peptide contained more elongated mitochondria (Figure 9A) compared to cells treated with BID BH3 peptide, whereby the mitochondria were smaller and appeared more spherical in structures (Figure 9B). Quantitative analysis was performed by measuring the aspect ratio (major axes divide by the minor axes) of the PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5646