BAX/BAK Independent Cell Death Figure 10. BH3 peptides induce an increase in Bcl-XL binding to dynamin-related protein 1 (DRP1). (A) Immunoblot of coimmunoprecipitation of BCL-XL and DRP1. Bax2/2/Bak2/2 MEFs overexpressing Flag BCL-XL were treated with DMSO, BID BH3 peptide (100 mM) or mutant BID BH3 peptide (100 mM). Flag antibody was used for immunoprecipitation. (B) Relative amounts of DRP1 binding to BCL-XL following treatment of mutant BID BH3 peptide (100 mM) or BID BH3 peptides (100 mM). BID BH3 peptide induced a two fold increase in DRP1 binding to BCL-XL. P value = 0.0122. Student’s unpaired t test was used to assess for statistical significance. (C) Bax2/2/Bak2/2 MEFs overexpressing DN DRP1 or empty pBabe construct as a control were treated for 1 hour with DMSO or indicated peptides. Percentage of cell death was determined by LDH release. Mean values6SEMs of 3 independent experiments are shown. (D) Wild Type MEFs overexpressing DN DRP1 or empty pBabe construct as a control were treated for 16 hours with DMSO or staurosporine (1 mM STS). Percentage of cell death was determined by assessing for PI positive cells by flow cytometry. Mean values6SEMs of 3 independent experiments are shown. (E) Bax2/2/Bak2/2 MEFs were pretreated with the mitochondrial fission inhibitor mdivi-1 (100 mM) for 30 minutes followed by 1 hour treatment with of BIM BH3 peptide (80 mM). Percentage of cell death was assessed by PI staining. Mean values6SEMs of 3 independent experiments are shown. (F) Wild type MEFs were pretreated with mdivi-1 (100 mM) for 30 minutes followed by 16 hour treatment with staurosporine (1 mM STS). Percentage of cell death was assessed by LDH release. Mean values6SEMs of 3 independent experiments are shown. doi:10.1371/journal.pone.0005646.g010 PLoS ONE | www.plosone.org 12 May 2009 | Volume 4 | Issue 5 | e5646