BAX/BAK Independent Cell Death Figure 3. Caspase inhibition does not inhibit BH3 Peptides induced cell death. (A) Bax2/2/Bak2/2 MEFs stably expressing a dominant negative caspase 9, or empty vector were treated with indicated peptides and after 1 hour the percentage of PI positive cells was analyzed by FACs analysis. Mean values6SEMs of 3 independent experiments are shown. (B) Bax2/2/Bak2/2 MEFs were treated with Q-VD-OPh (50 mM, broad range caspase inhibitor) for 1 hour prior to adding BID peptide (100 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. (C) Bax2/2/Bak2/2 MEFs expressing GAPDH, or control empty vector were treated with indicated peptides in the presence or absence of Q-VD-OPh (50 mM) for 1 hour. Percentage of PI positive cells was analyzed by flow cytometry. (D) Western blot demonstrating levels of GAPDH protein over-expression in Bax2/2/Bak2/2 MEFs. GAPDH was tagged to V5, therefore both endogenous and overexpressed levels could be identified. Empty vector was used as a transfection control. Bottom panel show tubulin levels, a loading control. (E) Bax2/2/Bak2/2 MEFs were treated with 3-Methyladenine (10 mM, autophagy inhibitor) for 1 hour prior to adding BID BH3 peptide (100 mM) or mutant BID BH3 peptide (100 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.g003 mitochondria. Statistical analysis showed that Bax2/2/Bak2/2 MEFs treated with BID BH3 peptide had a reduced aspect ratio compared to the mutant BID BH3 peptide (Figure 9C). These results further confirm that the mitochondria were indeed undergoing fragmentation, resembling that of mitochondrial fission. PLoS ONE | www.plosone.org 5 May 2009 | Volume 4 | Issue 5 | e5646