BAX/BAK Independent Cell Death Figure 8. Micro-injection of BH3 peptides induces mitochondrial fragmentation of Bax2/2/Bak2/2 MEFs. (A and B) Mitochondria of Bax2/2/ Bak2/2 MEFs were stained with Mitotracker Red CMXRos (50 nM) followed by microinjection of 100 mM BID (A) or mutant BID peptide (B) that does not contain 8D-arginines. Injected cells were tracked by dextran FITC. doi:10.1371/journal.pone.0005646.g008 a BAX/BAK dependent manner. By contrast, the expression of the BH3 only protein BAD which only binds a subset of pro- survival BCL-2 family proteins (BCL-2, BCL-XL, A1, BCL-w) does not induce cell death [30,31]. In accordance with these previous studies we also observe that expression of BIM protein and not BAD protein induces BAX/BAK dependent cell death (data no shown). In contrast, treating the cells with BIM BH3 peptide induces cell death even in the absence of BAX and BAK. Other BH3 peptides that can bind all the pro-survival proteins such as BID and PUMA can also induce cell death even in the absence of BAX and BAK. Additionally, neither the NOXA BH3 peptide, which only binds the pro-survival protein MCL-1, nor the BAD BH3 peptide, which binds only BCL-2, BCL-XL, A1 and BCL-w, failed to induce cell death. However, the combination of NOXA and BAD BH3 peptides did induce cell death in the absence of BAX/BAK. Furthermore, as controls throughout our study, we utilized when appropriate, either a mutant BID BH3 peptide or mutant BAD BH3 peptides. These peptides contain two amino acid mutated to alanine thus disrupting their binding to pro-survival BCL-2 family proteins [19,21]. The mutation still allowed for efficient transport of the peptides in cells. These controls indicate that the BH3 peptide killing of Bax2/2/Bak2/2 cells is not due non-specific effects such as the 8 D-arginine residues utilized to transport the peptides across cell membranes. Also, these observations in MEFs were corroborated utilizing a BAX/BAK deficient epithelial cell line. The BH3 peptides did not result in any detectable cytochrome c release from mitochondria isolated from Bax2/2/Bak2/2 cells or from intact Bax2/2/Bak2/2 cells. These peptides simply do not permeabilize membranes or else they would have released cytochrome c even in the absence of BAX/BAK. These results are consistent with previous findings that BH3 proteins or peptides require BAX/BAK for cytochrome c release [3,21]. However, the BH3 peptides resulted in depolarization of the mitochondrial PLoS ONE | www.plosone.org 10 May 2009 | Volume 4 | Issue 5 | e5646