BAX/BAK Independent Cell Death peptide (80 mM). Time lapse imaging was taken at 20 second intervals. Mean values6SEMs of 3 independent experiments are shown. (C) Bax2/2/ Bak2/2 MEFs treated for 1 hour with DMSO, BID BH3 peptide (100 mM), mutant BID BH3 peptide (100 mM), followed by 100 nM of calcein-AM labeling (and quenched with 0.4 mM Cobalt Chloride) in the presence or absence of ionomycin (500 nM). Mean calcein fluorescence was measured at 530 nm by flow cytometry. Results are expressed as mean values6SEMs of 5 independent experiments. (D) Bax2/2/Bak2/2 MEFs were treated with cyclosporine A (1 mM, CsA) and treated with BID BH3 peptide (100 mM), mutant BID BH3 peptide (100 mM) or BIM BH3 peptide (80 mM) for 1 hour. Percentage cell death was determined by assessing for PI staining by flow cytometry. Mean values6SEMs of 3 independent experiments are shown. (E) Bax2/2/Bak2/2 MEFs were treated with BAPTA-AM (5 mM) for 15 minutes prior to adding BID BH3 peptide (100 mM), mutant BID BH3 peptide (100 mM) or BIM BH3 peptide (80 mM) for 1 hour. Percentage cell death was determined by assessing for cell staining positive for propidium iodine by flow cytometry. Mean values6SEMs of 3 independent experiments are shown. doi:10.1371/journal.pone.0005646.g006 death upon overexpression of BH3 only proteins, the difference is that the BH3 peptides might bind to a larger pool of pro-survival BCL-2 family proteins. Alternatively, the BH3 peptides can perhaps access pro-survival proteins better than endogenous BH3 proteins. The BH3 peptides have been extensively utilized previously to probe both specificities of BH3 proteins binding to prosurvival BCL-2 proteins and the mechanism by which BH3 proteins induce release of cytochrome c [19–21,28]. Previous data indicate that expression of BH3 only protein BIM which can bind all the pro-survival BCL-2 family proteins can induce cell death in Figure 7. BH3 peptides induce mitochondrial fragmentation of Bax2/2/Bak2/2 MEFs. (A) Mitochondria of Bax2/2/Bak2/2 MEFs were stained with Mitotracker Red CMXRos (50 nM) and time lapse microcopy was performed. Pictures were taken prior to and at 1 minute intervals following treatment with 100 mM of mutant BID, BID and BIM BH3 peptides. (B) Time lapse microscopy of Bax2/2/Bak2/2 MEFs stained with Mitotracker RedCMXRos (50 nM). Cells were treated with sensitizer peptides, NOXA (75 mM) or BAD BH3 peptide (30 mM) alone, or in combination. The combination of NOXA (75 mM) and mutant BAD BH3 peptide (30 mM) was also analyzed. doi:10.1371/journal.pone.0005646.g007 PLoS ONE | www.plosone.org 9 May 2009 | Volume 4 | Issue 5 | e5646