by TMRE release. Mitochondria morphology was determined by Mitotracker CMX-ROS (Invitrogen) counterstain. Dynamic live cell imaging was performed on a Yokogawa spinning disc confocal fitted on a Nikon TE2000U microscope enclosed in 37C heated CO2 chamber, housed at the Northwestern University Cell Imaging Facility. Image acquisition was performed by Hama- matsu 9100C electron-multiplication CCD camera through a 1006 objective lens (N.A. 1.46). Care was taken during image acquisition to ensure that there were no saturated pixels. Image analysis was performed by MetaMorph (version 6.3r5) software. The release of TMRE upon membrane depolarization caused a decrease in intramitochondrial fluorescent intensity. To track the time-dependent TMRE fluorescent intensity changes, a region was drawn along the cell outline. This region was then transposed to a cell-free region in the same field of view adjacent to the cell being imaged. To obtain the mitochondrial/diffuse index, the intensity standard deviation [40] within these two regions was ratiome- trically compared over time as: (ICell 2 IBackground)/IBackground; wherein ICell represents the intensity standard deviation of within the region outlining the cell, and IBackground represents the intensity standard deviation within the exact same region transposed to a cell-free area. The mitochondria/diffuse index thus allowed us to simultaneously correct for background fluorescence fluctuations and track TMRE release. Mitochondrial Transition Pore Assay To assess for the mitochondrial transition pore opening, Bax2/2/ Bak2/2 MEFs were treated with corresponding BH3 peptides for 1 hour, trypsinized and labeled for flow cytometry using MitoPro- beTM Transition Pore Assay Kit (Invitrogen) according to manufac- ture’s protocol. The change in mean Calcein fluorescence of the mitochondria before and after addition of ionomycin indicates activation of the mitochondrial permeability transition pore. Mitochondria isolation and cytochrome c release Wild type or Bax2/2/Bak2/2 MEFs were collected in mitochondrial isolation buffer (250 mM sucrose, 10 mM Tris- Hcl pH 7.4, 0.1 mM EGTA. Mitochondria were obtained by mechanical cell disruption using a dounce homogenizer followed by 10 expulsions through a 27 gauge syringe and differential centrifugation. Isolated mitochondria were then re-suspended in experimental buffer (125 mM KCl, 10 mM Tris-MOPs, 5 mM glutamate, 2.5 mM malate, 1 mM KPO, 10 uM EGTA-Tris), and incubated with appropriate peptides for 30 minutes at room temperature. Cytochrome c release was assessed by ELISA using a mouse cytochrome c immunoassay from R& D Systems. As a control to assess for total (100%) cytochrome c release, isolated mitochondria were treated with 0.5% Tx-100 (Sigma) while the minimum value is cytochrome c release from cells treated with DMSO. Percentage cytochrome c release was calculated accord- ing to the following equation: %cyto c = (X-min cyto c)/(Total cyto c- min cyto c), whereby X is the value obtain for each experimental condition. Retroviral transfection Packaging cell line PLAT-E (kind gift of T. Kitamura) were transfected using Mirus TransIT Transfection reagent (Mirus Bio Corporation) according to the manufacturer’s protocol. 48 hours post transfection, medium containing virus was supplemented with 8 mg/ml polybrene (Sigma) for cell line infection and applied to MEFs. The pSiren vector (Clontech) was used to express short hairpin RNA (shRNA) sequences for Bok (59-CAGATCCGTCC- CAGCGTAT-39) and Drosophila melanogaster HIF (dHIF) (59- GCCTACATCCCGATCGATGATG-39). Cytochrome c GFP BAX/BAK Independent Cell Death construct was a kind gift from Dr. D.R. Green. DN-Drp-1 construct was a kind gift from Dr. R. Youle, which we re-cloned into pBabe GFP. Infected cells were selected with corresponding selection markers, and for GFP expressing cells, selection was achieved by sorting using the DakoCytomation MoFlo high speed multilaser droplet cell sorter at 488 nm. Electron Microscopy Treated cells were rinsed with PBS and fixed for 1 hour in 2.5% glutaraldehyde at room temperature followed by another rinse in 0.1 M cacodylate buffer. Coverslips were then incubated in secondary fixative of 2% osmium tetroxide for 30 minutes. Samples were then en-bloc stained with 3% uranyl acetate. Fixed cells were dehydrated in a graded series of ethanol and embedded in araldite and epon mixture. Following sectioning, the samples were contrasted with 6% uranyl acetate and Reynols lead. Images were taken on the JOEL 1220 Transmission Electron Microscope. Random cells were chosen for imaging and aspect ratio of all mitochondria within a cell section were calculated using the MetaMorph software (version 6.3r5). Immunoprecipitation Bcl-xL was immunoprecipitated using the FLAGH Tagged Protein Immunoprecipitation Kit (Sigma) per manufacturer’s protocol. Briefly, Bax2/2/Bak2/2 MEFs overexpressing FLAG- Bcl-xL were plated in 10 cm plates. At 50% confluency, cells were treated with 100 mM of BID or mutant BID BH3 peptide for 10 minutes. Cells were washed in PBS and Cell Lysis Buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% TRITONTM X-100) was added. Total protein was determined by Bradford Assay. Flag tagged Bcl-XL were immunoprecipiated with ANTI-FLAG M2 affinity gel. Equal protein was added to the gel and incubated overnight on a roller shaker at 4uC. Immunoprecipitates were separated on 12% poly- acrilimide gels and detected using anti-DRP1 antibody (H-300, Santa Cruz Biotechnology, 1:200) and Flag antibody (Sigma) as loading control. Microinjection Bax2/2/Bak2/2 MEFs were microinjected with BID or mutant BID peptide that does not contain 8D-arginine sequence. The peptides were dissolved in 10 mM Tris and Dextran FITC was added as a marker for identification of injected cells. Microinjec- tion was carried out on an Axovert 135 microscope equipped with an Ependorf Femtojet microinjector. Cells were injected using Femtotip II injection capillary at a pressure of 22 hPa. Injected cells were viewed 30 minutes following injection using a fluorescence microscope LSM510 and pictures were captured using a 636 lens objective. Other reagents MDIVI compound was purchased from Ryan Scientific and was resuspended in DMSO. 3-Methyladenine, staurosporine, and cyclosporin A were purchased from Sigma Aldrich. Q-VD-OPh was purchased from R&D Scientific. Mitotracker CMX-ROS and tetramethylrhodamine ethyl ester (TMRE) were purchased from Invitrogen. Supporting Information Figure S1 FITC tagged peptide uptake in Bax2/2/Bak2/2 MEFs. (A) Bax2/2/Bak2/2 MEFs treated with DMSO, (B) 100 mM mutant BID or (C) 100 mM BID BH3 peptides that were PLoS ONE | www.plosone.org 14 May 2009 | Volume 4 | Issue 5 | e5646