Bid-induced mitochondrial membrane permeabilizationwaves propagated by local reactive oxygen species(ROS) signalingCecilia Garcia-Perez1, Soumya Sinha Roy1,2, Shamim Naghdi, Xuena Lin, Erika Davies, and György Hajnóczky3

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107

Edited by Tullio Pozzan, University of Padova, Padova, Italy, and approved February 7, 2012 (received for review November 9, 2011)

Bid-induced mitochondrial membrane permeabilization and cyto-chrome c release are central to apoptosis. It remains a mysteryhow tiny amounts of Bid synchronize the function of a large num-ber of discrete organelles, particularly in mitochondria-rich cells.Looking at cell populations, the rate and lag time of the Bid-in-duced permeabilization are dose-dependent, but even very lowdoses lead eventually to complete cytochrome c release. By con-trast, individual mitochondria display relatively rapid and uniformkinetics, indicating that the dose dependence seen in populationsis due to a spreading of individual events in time. We report thatBid-induced permeabilization and cytochrome c release regularlydemonstrate a wave-like pattern, propagating through a cell ata constant velocity without dissipation. Such waves do not dependon caspase activation or permeability transition pore opening.However, reactive oxygen species (ROS) scavengers suppressedthe coordination of cytochrome c release and also inhibited Bid-induced cell death, whereas both superoxide and hydrogen perox-ide sensitized mitochondria to Bid-induced permeabilization. Thus,Bid engages a ROS-dependent, local intermitochondrial potentia-tion mechanism that amplifies the apoptotic signal as a wave.

wave propagation | intermitochondrial signaling | calcium |outer mitochondrial membrane | cell death

Bid, a proapoptotic Bcl-2 family protein, induces apoptosis bymediating the release of cytochrome c (cyto c) and other

proteins from mitochondria to the cytosol. Both the appearance ofmitochondrial proteins in the extramitochondrial space andthe loss of mitochondrial integrity can induce multiple mechanismsto execute apoptosis (1). In Bid-deficient mice, Fas-, granzyme B-,and heat shock/caspase-2–induced apoptosis are impaired (2–4).“Death” receptors (TNFR1/Fas) engage caspase-8, whereas vari-ous stress conditions induce calpain, caspases, cathepsins, or gran-zyme B to cleave and activate Bid (5–8). Truncated Bid (tBid) bindsto the outer mitochondrial membrane (OMM) to induce Bak/Bax-dependent release of the soluble intermembrane space (IMS)proteins (9). It is debated whether tBid directly activates theproapoptotic Bak/Bax (10–13) or rather engages and antagonizesthe function of their prosurvival Bcl-2 relatives (14). tBid was alsoreported to interact with cardiolipin (11, 15, 16), Mtch-2 (17, 18),and voltage dependent anion-selective channel in the OMM (19).The predominant fraction of cyto c and other IMS proteins

are compartmentalized in the cristae, produced by the foldings ofthe inner mitochondrial membrane (IMM) (20), and is bound tocardiolipin (21). Remodeling of the cristae may occur via a pro-cess that involves a cyclosporine A (CsA)-sensitive factor anddisruption of Opa1 oligomers (22, 23). The binding of cyto c tocardiolipin is disrupted by oxidative stress (24). Despite all of theobstacles, tBid induces rapid discharge of the entire cyto c poolof hundreds to thousands of discrete mitochondria in each cell.In various cell types treated with tBid-linked agonists, once ini-tiated, cyto c release from all mitochondria is completed within≈5 min (25, 26). Exposure of permeabilized cells to maximal tBid(≥15 pmol/mg protein) causes complete release of cyto c andSmac in 3 min (27). However, the concentration of endogenous

Bid is much lower, indicating the need for an auxiliary mecha-nism in tBid-induced mitochondrial membrane permeabilization.This mechanism would need to be particularly effective in cellsabundant in mitochondria, such as muscle or hepatic cells. tBidmay be assisted by other cytoplasmic factors (e.g., Bax). Alter-natively, mitochondria undergoing tBid-induced membrane per-meabilization may sensitize adjacent mitochondria to tBid. Toaddress this possibility, the temporal and spatial pattern of tBid-induced mitochondrial membrane permeabilization was resolvedin real time and at the level of single mitochondria.

Results and DiscussionCyto c release and the ensuing mitochondrial depolarization wereinduced by tBid in a dose- and time-dependent manner in sus-pensions of permeabilized hepatic (HepG2) (Fig. 1 A and B) (27)and cardiac muscle-derived (H9c2) cell lines (Fig. S1 A and B).Dose dependency was apparent for the rate and lag time of thetBid-induced permeabilization (Fig. 1A and Fig. S1A). However,over a broad range of tBid concentrations (0.5–50 nM), cyto c re-lease, adenylate kinase release, and depolarization were progressive(Fig. 1B), and even very low doses lead eventually to complete re-lease (Fig. S1A). By contrast, submaximal digitonin, a cholesterol-selective detergent, caused rapid and partial cyto c release andmitochondrial depolarization, which reached their peak in 1 min(Fig. 1A). The digitonin dose determined the incremental magni-tude of the response (Fig. S1C). This behavior indicates a simplestoichiometric relationship between the concentration of detergentand the amount of mitochondrial membrane it is able to per-meabilize, in contrast to tBid, which activates progressive release ofIMS proteins in populations of permeabilized cells.In real-time imaging studies, single permeabilized H9c2 cells

transfected with cyto c–GFP also showed gradual development oftBid-induced cyto c–GFP release (Fig. 1C). Cyto c release startedat 2.5–7.5 min after submaximal tBid addition and involved allmitochondria (Fig. 1C). Individual cells displayed different lagtimes and rate of release (Fig. 1C). For single whole cells, the lagtime of cyto c–GFP release was inversely proportional, and therate directly related to tBid concentration across a broad range(2–100 nM) (Fig. S2). Eventually, the entire cyto c–GFP pool wasmobilized, even if submaximal tBid was used. Confocal imaging ofH9c2 cells expressing both cyto c–GFP and mitochondrial matrix-

Author contributions: C.G.-P., S.S.R., S.N., X.L., and G.H. designed research; C.G.-P., S.S.R.,S.N., X.L., E.D., and G.H. performed research; C.G.-P., S.S.R., S.N., X.L., E.D., and G.H.analyzed data; and C.G.-P., S.S.R., and G.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1C.G.-P. and S.S.R. contributed equally to this work.2Present address: Institute of Genomics and Integrative Biology, Council of Scientific andIndustrial Research, New Delhi, India.

3To whom correspondence should be addressed. E-mail: Gyorgy.Hajnoczky@jefferson.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1118244109/-/DCSupplemental.

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targeted DsRed showed in each cell >100 discrete mitochondriaof either globular or tubular shape and some mitochondrialclusters (Fig. 1D). Individual mitochondria displayed distinct lagtime but showed uniform, single-step kinetics for tBid-inducedcyto c–GFP release (Fig. 1D). Thus, nonsynchronous and com-plete cyto c release from individual mitochondria underlies thecontinuous progression of the release as measured in whole cellsand in cell populations. If tBid is sufficient to activate cyto c re-lease from a subset of mitochondria, then the whole population ofmitochondria goes along.In the experiments described above, tBid was present in the

cytosol during the entire course of OMM permeabilization. Totest whether continuing tBid recruitment is required for per-meabilization of the whole mitochondrial population, suspen-sions of skinned cells were pretreated with a low dose of tBid (0.5nM for 100 s), which was washed out before noticeable cyto crelease occurred. Immunoblotting of the membrane and cytosolfractions for tBid revealed detectable tBid binding to the mito-chondria, but the remaining large cytosolic fraction was effectivelyremoved by the washout, preventing further tBid recruitment tothe OMM. Washout of tBid only partially suppressed the pro-gression of the cyto c discharge and ensuing depolarization duringsubsequent incubation (Fig. S3). This delayed response was alsoinsensitive to caspase inhibitors, to Ca2+ chelators, and to CsA

(Fig. S3). Complementing these results, in single permeabilizedcells, washout of tBid failed to prevent completion of cyto c–GFPrelease (n = 3).Collectively, these results suggest that progression of the tBid-

induced OMM permeabilization would not depend on continu-ous tBid recruitment, caspase activation, Ca2+, or mitochondrialpermeability transition pore (PTP) opening. Notably, the cyto-plasm was also removed in the tBid washout experiments, in-dicating that continuous recruitment of a soluble factor from thecytosol is also needless for the progression of tBid-inducedOMM permeabilization. A recent article described waves of cy-toplasm-to-mitochondria Bax transfer (28), but this could notoccur here, and the tBid-induced OMM permeabilization wasprobably mediated by Bak or Bax constitutively present in themitochondria (29). Although the washout experiment alone doesnot exclude the possibility that slow activation of the Bak/Baxeffector mechanism by tBid sets the tempo for the progress ofthe cyto c release, this mechanism would not result in a regen-erative cyto c release wave (see below).As an alternative mechanism for the tBid-induced all-or-none

response, we considered that mitochondria undergoing per-meabilization may provide some factors to facilitate the per-meabilization of the rest of the organelles. An initial support tothis idea came from the experiments in which the time course of

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Fig. 1. Temporal and spatial organization of the tBid-induced cyto c release. (A) Distinct time- and dose-dependence of tBid- and digitonin-induced mi-tochondrial permeabilization in cell suspensions. ΔΨm was monitored in skinned permeabilized HepG2 cells in the presence of 2 mM succinate, 2 mM ATP, and5 μg/mL oligomycin. Under these conditions cyto c depletion of the mitochondria causes depolarization. Cyto c release and the ensuing depolarization evokedby various concentrations of digitonin and tBid. For the cyto c determination, cytosol and membrane samples were separated by centrifugation at 550 s. (B)Continuous cyto c release, adenylate kinase release, and ensuing ΔΨm loss evoked by submaximal tBid in suspensions of skinned HepG2 cells. Arrows indicatethe time points at which cytosolic samples were generated by rapid filtration of the cells for quantitation of adenylate kinase and cyto c release. (C) Completerelease of cyto c–GFP elicited by tBid in single cells. The gray images show the cyto c–GFP fluorescence (Upper) in three skinned H9c2 cells challenged with2 nM tBid. The red overlays show the fluorescence change at each time point and thus represent cyto c–GFP release. The graph illustrates the normalized cytoc–GFP release responses for single cells (in red showing the cells from the images) and the mean response for 15 cells (black line). The horizontal line shows thecell-to-cell variability in the time when the half-maximal response was attained. (D) Kinetics of cyto c–GFP release from single mitochondria. Images show themerge of cyto c–GFP fluorescence (green) and mtRed fluorescence (red) images for a whole H9c2 cell (Upper Left) and for a subcellular region (white box) atselected time points during exposure to tBid (10 nM added at 75 s). Because breakdown of the OMM barrier leads to loss of only the green fluorescence, cytoc–GFP release appears as a yellow to red color shift in the images. The graphs show the time course of cyto c–GFP release for individual mitochondria markedby numbers (Left). (E) Possible role of local interactions between adjacent mitochondria in the cyto c–GFP release. The initial rate of the tBid (5 nM)-inducedcyto c–GFP release in regions of high (densely populated) and low (like the boxed area in D) mitochondrial density, separately.

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cyto c–GFP was examined in subcellular regions of differentmitochondrial densities, and areas of high mitochondrial densityexhibited steeper cyto c–GFP release than the regions of lowmitochondrial density (Fig. 1E). However, the key evidence in-dicating a local coupling between adjacent organelles undergoingOMM permeabilization came from closer observation of thespatial organization of cyto c–GFP release in cells densely

populated by mitochondria. In permeabilized differentiatingH9c2 myoblasts, the cyto c–GFP release started at discrete sitesand propagated as a wave over several-hundred-micrometerdistances at full strength in a few minutes (Fig. 2A and MovieS1). Similar results were obtained in permeabilized primary hu-man cardiac muscle (HCM) cells (Fig. 2B). The waves displayedconstant velocity (H9c2: 0.19 ± 0.01 μm/s, n = 91; HCM: 0.31 ±

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Fig. 2. tBid-induced cyto c–GFP release waves in per-meabilized cells. (A) Fluorescence image time series showsthe spatial pattern of cyto c–GFP release evoked by tBid(5 nM) in a skinned differentiating H9c2 myoblast. A line-scan image was created by selecting a line parallel to thedirection of wave propagation, obtaining the correspondingfluorescent signal from every image in the time series, andstacking the successive lines horizontally. This presentationshows that the rate of wave spreading was constantthroughout the cell. The graph shows corresponding tracesof fluorescence calculated for three regions of the cell alonga line transverse to the apparent wave front. (B) The cyto c–GFP release wave induced by tBid (5 nM) a permeabilizedprimary HCM cell is shown by the red overlays that mark thelocation of the fluorescence change at each time point.

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Fig. 3. ROS-dependence of the spatiotemporal organiza-tion of the tBid-induced cyto c–GFP release. (A) Temporalcoordination of cyto c–GFP release in H9c2 cells pre-incubated with zVADfmk (50 μM) to suppress caspase acti-vation, with EGTA (2 mM) to remove Ca2+, in the absence ofsubstrates and rotenone (10 μM) to minimize ROS forma-tion, and with MnTMPyP (250 μM for 10 min) is shown asthe average time elapsed between 10% and 90% release ineach cell. (B) Cyto c-GFP release in cells pretreated withMnTMPyP or solvent. Single-cell and mean (thick line) ki-netics are shown for an experiment (Left). For each cell thetime required to attain 10%, 50%, and 90% cyto c–GFPrelease was measured, and the average responses wereplotted (Right). (C) Propagation rate for control andMnTMPyP-pretreated cells exhibiting cyto c–GFP releasewaves. (D) Matrix superoxide levels in cyto c–GFP express-ing, permeabilized cells loaded with Mitosox Red. A gradualmitochondrial superoxide generation induced by tBid (37nM, Upper) closely attended cyto c–GFP release (Lower) isshown as the mean responses of all cells. Antimycin A (Ant.A, 5 μM), a complex III inhibitor, was used as a positivecontrol for mitochondrial ROS generation. (E) Spatiotem-poral relationship between matrix superoxide increase andcyto c–GFP release. Records were obtained as described forD. The gray images show the cyto c–GFP fluorescence (Up-per) and MitoSox Red fluorescence (Lower) in individualpermeabilized cells challenged with tBid (35 nM). The redand cyan overlays show the fluorescence change at eachtime point for cyto c–GFP and MitoSox, respectively, andthus represent the spatial localization of the cyto c releaseand superoxide increase within the cells. The temporal lo-calization is illustrated by the graphs for three selected cells.Cyto c-GFP release spread as a wave along the long axis ofcell 2, allowing assessment of the spatial pattern of thecorresponding superoxide increase (2a and 2b).

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0.05 μm/s, n = 10) and a stable kinetic of release in each region(Fig. 2). This indicated that the cyto c–GFP release waves aresupported by a regenerative mechanism that could result froma local coupling between adjacent mitochondria.We next searched for the trigger that sets off this wave. The

spatiotemporal coordination of tBid-induced cyto c–GFP releasewas not altered by zVADfmk or EGTA (Fig. 3A) or rutheniumred or CsA (Fig. S4A). Recently, death receptor-induced apo-ptosis has been shown to involve Bid-dependent reactive oxygenspecies (ROS) formation (30). We found that incubation of thecells under conditions that limit the mitochondrial ROS pro-duction (no substrate + rotenone) along with MnTMPyP, a ROSscavenger, interfered with the coordination of tBid-induced cytoc–GFP release (Fig. 3 A and B). The propagation rate of the cytoc–GFP release waves was less in the presence of MnTMPyP (Fig.3C). Overexpression of a mitochondrial superoxide dismutase,SOD2, also slowed down the tBid-induced cyto c–GFP releasewaves (Fig. S4B). These results implicated ROS in the pro-gression of tBid-induced cyto c release.Mitochondrial ROS (superoxide) during cyto c release was

measured in cells expressing cyto c–GFP and loaded withMitoSox. tBid caused a gradual ROS increase closely coupledwith cyto c–GFP release (Fig. 3D). Because the MitoSox fluo-rescence was weak relative to the spectrally overlapping cellularautofluorescence, the spatial distribution of prestimulation ROScould not be correlated with the propagation pattern of the cytoc–GFP release waves. However, time-lapse images showed thatthe ROS increase slightly preceded cyto c–GFP release in eachcell and showed a spatial pattern similar to that of cyto c–GFPrelease in cells exhibiting cyto c release waves (Fig. 3E). Takentogether, these results have provided some clues that tBid stim-ulates mitochondrial ROS production to coordinate the per-meabilization of subsets of mitochondria.

A potential mechanism for ROS to support regeneration of tBid-induced cyto c release is to enhance the sensitivity of mitochondriatoward tBid-induced OMM permeabilization. Consistent with thisidea, the cyto c release and the ensuing depolarization evoked bysubmaximal tBid was suppressed by MnTMPyP in suspensions ofpermeabilized H9c2 cells (Fig. 4A, Upper). Similarly, attenuatedcyto c release and depolarization in response to suboptimal tBidwas recorded in cells overexpressing SOD2 (Fig. 4A, Lower).Furthermore, exogenously provided ROS (both superoxide andH2O2) sensitized mitochondria toward tBid (Fig. 4B). Notably,the given ROS by itself could not evoke any cyto c release, in-dicating that tBid also had to be present, most likely on everymitochondrion. The ROS-induced sensitization did not affect thebinding of tBid to the mitochondria (Fig. S5A) but promoted theBak-oligomerization elicited by tBid (Fig. S5B). Thus, ROSproduced during tBid exposure can support wave propagation byenhancing the sensitivity of the mitochondria to tBid-inducedpermeabilization. The relevant target of ROS is between tBidbinding and Bak oligomerization.Finally, we tested the presence and relevance of tBid-depen-

dent cyto c release waves in intact cells. First, cyto c–GFP releasewaves were recorded in H9c2 cells infected with tBid adenovirus(Fig. 5A). Subsequently, cells were pretreated with TNF-α pluscycloheximide to activate the Bid pathway through death re-ceptors. Imaging of the cells was performed 14–19 h aftertreatment. Cyto c-GFP release waves spreading with stable ve-locity could be discerned in almost 25% of the cells that un-derwent cell death. However, waves probably occurred in morecells but remained undetected because during the several-hour-long recordings it was not possible to use high temporal or spatialresolution. Importantly, the velocity of the waves (0.23 ± 0.02μm/s, n = 18) was very similar to that of tBid-induced waves inpermeabilized cells. Furthermore, for the TNF-α/tBid-inducedwaves, the propagation velocity was lower than that for the

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Fig. 4. ROS-dependence of cyto c release by tBidin suspensions of skinned H9c2 myoblasts. (A)Suppression of tBid-induced cyto c release andΔΨm loss by MnTMPyP (250 μM, Left) and over-expressed SOD2 (Right). (B) Potentiation of tBid (1nM added at 300 s)-induced cyto c release (Lower)and ΔΨm loss (Upper) by pretreatment with H2O2

(60 μM) (Center) or with xanthine (X, 60 μM) andxanthine oxidase (XO, 6 mU/mL), a superoxideanion generating system (Right).

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Ca2+-induced PTP-dependent IMM permeabilization waves (1μm/s) (31), and the ΔΨm decreased only slowly and graduallyafter cyto c–GFP release, indicating that the IMM barrier wasmaintained (Table S1). When MnTMPyP was included in thebuffer, fewer cells died, fewer cyto c–GFP release waves wereobserved, and the remaining waves showed greatly reduced prop-agation velocity (Fig. 5B, bar charts, 0.03 ± 0.01 μm/s, n = 3).Complementing these results, MnTMPyP effectively inhibitedthe ability of tBid to induce apoptosis (Fig. 5C).Collectively, these results indicate that tBid-induced cyto c

release uses an intermitochondrial amplification mechanism topropagate cyto c release and to establish an all-or-none behaviorof OMM permeabilization. We propose that the mitochondriamost sensitive to tBid produce some ROS that sensitizes theirneighbors toward tBid to facilitate their recruitment to theOMM permeabilization (Fig. 5D). Recent evidence supports thattBid binds to cardiolipin (11, 15, 16) and involves ROS formation(30, 32) to dislodge cyto c bound to cardiolipin (24). This se-quence provides a potentiation mechanism for cyto c releasefrom a single mitochondrion. However, the present data implythat this potentiation mechanism may also couple adjacent mi-tochondria, providing a spatial amplification mechanism for cytoc release propagation. ROS microdomains (33) might promotethe translocation of cytoplasmic proapoptotic factors to theOMM [e.g., Bax (28)]. However, Bid-dependent cyto c releasewaves also occurred in permeabilized cells, where the cytosol wasomitted. Furthermore, the PTP, which is commonly involved inpropagation of mitochondrial activity by Ca2+ (31, 34–38) orROS (39) also seems dispensable for tBid-induced cyto c release.In addition, ROS can promote its own production (40) and

induce regenerative IMM permeabilization by itself (39, 41), buthere an interplay between Bid and relatively low levels of ROSseems to provide the wave propagation mechanism. Potentiationof tBid-induced Bak oligomerization by ROS indicates that ROSproduced and released by the mitochondria most sensitive to tBidmight sensitize tBid-induced Bak activation in the adjacent or-ganelles. Because in the cell type used in this study we havepreviously demonstrated Ca2+-induced and PTP-dependent IMMpermeabilization waves (31), multiple regenerative mechanismslikely exist in a single cell type. The differences between Ca2+-and tBid-induced waves are listed in Table S1. Our data alsosuggest that spreading of cyto c release by mitochondrial waves isrelevant for orderly execution of apoptosis. Both the most fa-vorable conditions and the biggest need for local inter-mitochondrial amplification mechanisms are presented by largecells abundant in mitochondria. Intriguingly, the interplay be-tween tBid and ROS may be important for tissue injury attrib-uted to an enhanced ROS formation [e.g., ischemia-reperfusioninjury (42, 43)] and could also be a novel target for therapeuticapoptosis induction.

MethodsCell Culture and Transfection.HepG2 and H9c2 cells were cultured as describedbefore (27, 31). HCM were obtained from ScienCell and were maintainedusing the manufacturer’s protocol. Cells were transfected with plasmid DNAas described earlier (31). tBid adenoviruses were produced and used aspreviously described (17). SOD2 adenovirus infection was done for 24–36 husing 2,000 viral particles per cell. Viral transduction resulted in expressionof tBid in 80% ± 4% of the cells (n = 4) and a substantial increase in the totalamount of tBid (Fig. 5A).

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Fig. 5. Cyto c-GFP release waves and ensuing cell death in intact H9c2 cells. (A) Cyto c-GFP release wave in a tBid adenovirus infected cell recorded ≈11 h afterinfection (Left). Verification of tBid expression and the ensuing cyto c release (Right). Samples: Cells infected with tBid (tBid-Ad) or control (Ad) adenovirusand noninfected control (mock), and recombinant tBid (tBid). (B) Cyto c-GFP release waves evoked by 15 h treatment with TNF-α (20 nM) + cycloheximide(CHX, 4 μg/mL). The line-scan image shows that the rate of wave spreading was constant throughout the cell. The bar charts shows the TNF-α–induced cellkilling, cyto c–GFP release wave frequency and wave velocity obtained in the absence and presence of MnTMPyP (50 μM) (n = 278, 36, 64, 3, 18, and 3,respectively; *P < 0.001). (C) MnTMPyP provides protection against tBid adenovirus-induced killing of H9c2 myoblasts. Flow cytometry analysis was conducted14 h after infection with tBid adenovirus or control adenovirus (n = 3). (D) Scheme showing the proposed propagation mechanism of tBid-induced waves ofOMM permeabilization.

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ΔΨm, Cyto C, and Adenylate Kinase Assay in Suspension of Permeabilized Cells.Measurement of ΔΨm in permeabilized cells (2.4 mg protein) was done asdescribed previously (27, 29). Recombinant full-length Bid and caspase-8-cleaved Bid (tBid) were produced as previously described (44) or purchasedfrom R&D Systems. At the end of the fluorimetric measurements of ΔΨm,cytosol was separated from the membranes by centrifugation at 10,000 × gfor 5 min or by rapid filtration (27). Supernatant or membrane proteins wereresolved in 12% (wt/vol) SDS-polyacrylamide gel. Immunoblotting was car-ried out using a cyto c (Pharmingen) or a Bid antibody (R&D Systems).Adenylate kinase activity was measured using a coupled optical enzymeassay (45).

Live Cell Microscopic Imaging. Before use, the cells were preincubated for 20min in an extracellular medium containing 2% (wt/vol) BSA at 37 8C. Formeasurements of ΔΨm the cells were loaded with 25–50 nM tetrame-thylrhodamine ethyl ester (TMRE) for 20 min at room temperature, and 2–5nM TMRE was added during the recordings. For mitochondrial ROS meas-urements, cells were loaded with 10 μM MitoSox Red (Invitrogen) for 20 minat 37 8C. Intact cell measurements were performed in an extracellular me-dium containing 0.25% (wt/vol) BSA at 35 8C. For imaging of mitochondria inskinned cells, the samples were prepared as described earlier (31). Fluores-cence imaging was performed using an inverted microscope (Leica DMIRE2;40×) fitted with a cooled CCD camera (Photometrics) and a computer con-trolled motorized turret, allowing us to alternate among optical filter setsfor fluorescein (for cyto c–GFP) and for rhodamine [for TMRE, MitoSox Red,and propidium iodide (PI)] with a 6-s (ΔΨm) and 30-s (ROS) acquisition delay.

Confocal imaging experiments were performed using a BioRad Radiancesystem fitted to an Olympus IX70 microscope or a Zeiss LSM780 system usinga 40× objective. The laser source was used for imaging of cyto c–GFP at ex:488 nm and mtRed at ex: 568 nm or 561 nm. All image analysis was done inSpectralyzer imaging software.

Flow Cytometry. H9c2 cells were treated with MnTMPyP (50 μM added 2 hbefore infection) and infected with tBid adenovirus for 14 h. The concen-tration of MnTMPyP was lowered in these studies because 150–250 μMMnTMPyP caused increased cell killing by itself during prolonged treatment.Then the cells (both attached and detached) were harvested and washedonce with PBS. Cell pellets were incubated with Annexin-V Alexa Fluor 488conjugate (1:40 dilution; Invitrogen) and PI (2.5 μg/mL) at room temperaturein dark for 15 min. Samples were analyzed within 1 h by a flow cytometer(488 nm and 568 nm excitation).

Statistics. Experiments were carried out with three or more different cellpreparations. Data are presented as means ± SE. Significance of differencesfrom the relevant controls was calculated by Student t test.

ACKNOWLEDGMENTS. We thank Drs. Bruno Antonsson (Serono PRI), AtanGross (Weizmann Institute), and Muniswamy Madesh (Temple University) forproviding us with recombinant tBid, tBid, and SOD2 adenoviruses, respec-tively, and Drs. Tamas Balla, Atan Gross, Jan Hoek, Suresh K. Joseph, andMr. David Weaver for comments on the manuscript. This work was supportedby National Institutes of Health Grant GM59419 (to G.H.).

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