Regulation of the transcription factor EB-PGC1α axis by beclin-1 controls mitochondrial quality and

23 Pages·2015·10.78 MB·English

Washington University School of Medicine
Digital Commons@Becker
Open Access Publications
2015
Regulation of the transcription factor EB-PGC1α
axis by beclin-1 controls mitochondrial quality and
cardiomyocyte death under stress
Xlucul Ma
Washington University School of Medicine in St. Louis
Halyan Liu
Washington University School of Medicine in St. Louis
John T. Murphy
Washington University School of Medicine in St. Louis
Sarah R. Foyil
Washington University School of Medicine in St. Louis
Rebecca J. Godar
Washington University School of Medicine in St. Louis
See next page for additional authors
Follow this and additional works at:http://digitalcommons.wustl.edu/open_access_pubs
Recommended Citation
Ma, Xlucul; Liu, Halyan; Murphy, John T.; Foyil, Sarah R.; Godar, Rebecca J.; Abulrqeba, Haedar; Weinheimer, Carla J.; Barger, Philip
M.; and Diwan, Abhinav, ,"Regulation of the transcription factor EB-PGC1α axis by beclin-1 controls mitochondrial quality and
cardiomyocyte death under stress." Molecualar and Cellular Biology.35,6. 956-976. (2015).
http://digitalcommons.wustl.edu/open_access_pubs/3964
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Authors
Xlucul Ma, Halyan Liu, John T. Murphy, Sarah R. Foyil, Rebecca J. Godar, Haedar Abulrqeba, Carla J.
Weinheimer, Philip M. Barger, and Abhinav Diwan
This open access publication is available at Digital Commons@Becker:http://digitalcommons.wustl.edu/open_access_pubs/3964
(cid:2)
Regulation of the Transcription Factor EB-PGC1 Axis by Beclin-1
Controls Mitochondrial Quality and Cardiomyocyte Death under
Stress
XiucuiMa,a,cHaiyanLiu,a,cJohnT.Murphy,aSarahR.Foyil,a,cRebeccaJ.Godar,a,cHaedarAbuirqeba,aCarlaJ.Weinheimer,a
PhilipM.Barger,aAbhinavDiwana,b,c
DivisionofCardiologyandCenterforCardiovascularResearch,DepartmentofInternalMedicine,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,USAa;
DepartmentofCellBiologyandPhysiology,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,USAb;JohnCochranVAMedicalCenter,St.Louis,Missouri,
USAc
Incardiacischemia-reperfusioninjury,reactiveoxygenspecies(ROS)generationandupregulationofthehypoxia-induc-
ibleproteinBNIP3resultinmitochondrialpermeabilization,butimpairmentinautophagicremovalofdamagedmito-
chondriaprovokesprogrammedcardiomyocytedeath.BNIP3expressionandROSgenerationresultinupregulationof
beclin-1,aproteinassociatedwithtranscriptionalsuppressionofautophagy-lysosomeproteinsandreducedactivationof
transcriptionfactorEB(TFEB),amasterregulatoroftheautophagy-lysosomemachinery.Partialbeclin-1knockdown
transcriptionallystimulateslysosomebiogenesisandautophagyviamTORinhibitionandactivationofTFEB,enhancing
removalofdepolarizedmitochondria.TFEBactivationconcomitantlystimulatesmitochondrialbiogenesisviaPGC1(cid:2)in-
ductiontorestorenormallypolarizedmitochondriaandattenuateBNIP3-andhypoxia-reoxygenation-inducedcelldeath.
Conversely,overexpressionofbeclin-1activatesmTORtoinhibitTFEB,resultingindeclinesinlysosomenumbersand
suppressionofPGC1(cid:2)transcription.Importantly,knockdownofendogenousTFEBorPGC1(cid:2)resultsinacompleteor
partialloss,respectively,ofthecytoprotectiveeffectsofpartialbeclin-1knockdown,indicatingacriticalroleforbothmi-
tochondrialautophagyandbiogenesisinensuringcellularviability.Thesestudiesuncoveratranscriptionalfeedbackloop
forbeclin-1-mediatedregulationofTFEBactivationandimplicateacentralroleforTFEBincoordinatingmitochondrial
autophagywithbiogenesistorestorenormallypolarizedmitochondriaandpreventischemia-reperfusion-inducedcardio-
myocytedeath.
Preservation of healthy mitochondria is essential for energy todrivereformationofnewlysosomes(18–20).Thisisfacili-
generationandmaintenanceofcontractilefunctionincar- tated via a transcriptional induction of autophagy-lysosome
diacmyocytes(1).Incardiacischemia-reperfusion(IR)injury, machinery proteins orchestrated by nuclear translocation of
mitochondrial permeabilization results in activation of pro- the basic helix-loop-helix (bHLH) transcription factor EB
grammed cell death pathways and cardiomyocyte loss (2). (TFEB)(21–25),amasterinduceroftheautophagy-lysosome
Removal of damaged mitochondria by macroautophagy, a machinery (23), thereby sustaining autophagic flux. In con-
lysosomaldegradativepathway,isessentialtopreventcardiomy- trast,lysosomenumbersprogressivelydeclinewithBNIP3-in-
ocytedeathandlimitmyocardialinfarctsize(3,4).Cardiomyo- ducedautophagy,withoutreplenishment(15),suggestingthat
cyteautophagyisupregulatedwithIRinjury(5),butautophago-
animpairmentinthistranscriptionalresponseengenders“in-
some processing is impaired early after reperfusion, which
sufficient”cytoprotectiveautophagy.
preventsautophagicremovalofdamagedmitochondria(6).The
Relevant to this discussion is our observation that upon
hypoxicinsultalsoprovokestranscriptionalinductionofBNIP3
reperfusion/reoxygenation, a rapid reactive oxygen species
(Bcl2andnineteen-kilodaltoninteractingprotein3),aprodeath
(ROS)-induced increase in beclin-1 abundance paradoxically
Bcl2familyprotein(7,8)whichistargetedtoandpermeabilizes
impairs autophagic flux in cardiomyocytes (6). Interestingly,
mitochondria(9–11)andtriggerscardiomyocytedeathinIRin-
whilebasalbeclin-1levelsplaycriticalrolesinautophagosome
jury(12).WhileBNIP3hasbeensuggestedtofacilitatemitochon-
drialautophagybyfunctioningasanadaptortosequesterdam-
aged mitochondria within autophagosomes (13, 14), increased
BNIP3expressionprovokesdeclinesinlysosomenumbers,with Received25August2014 Returnedformodification15September2014
Accepted30December2014
impairedautophagicflux,resultinginaccumulationofdamaged
Acceptedmanuscriptpostedonline5January2015
mitochondriaandcardiomyocytedeath(15).Theseobservations
CitationMaX,LiuH,MurphyJT,FoyilSR,GodarRJ,AbuirqebaH,WeinheimerCJ,
implicateafailureoftheautophagy-lysosomemachinerytoclear BargerPM,DiwanA.2015.RegulationofthetranscriptionfactorEB-PGC1(cid:2)axisby
damagedmitochondriaasacauseofcelldeathwithIRinjury,but beclin-1controlsmitochondrialqualityandcardiomyocytedeathunderstress.
theunderlyingmechanismsremaintobedefined. MolCellBiol35:956–976.doi:10.1128/MCB.01091-14.
Mitochondria are also targeted for degradation by starva- AddresscorrespondencetoAbhinavDiwan,[email protected].
tion, wherein autophagy is critical for cell survival (16, 17). Copyright©2015,AmericanSocietyforMicrobiology.AllRightsReserved.
Interestingly,withstarvation,lysosomenumbersrapidlyplum- doi:10.1128/MCB.01091-14
met(18),butendogenousmechanismsarepromptlyrecruited
956 mcb.asm.org MolecularandCellularBiology March2015 Volume35 Number6
Beclin-1RegulatesMitochondrialQualityviaTFEB
formation(26)andprotectionagainstcardiomyocytedeath(6),we Generationofadenoviralconstructs.Lentiviralparticlescodingfor
observedthatincreasedbeclin-1abundanceissufficienttosuppress mCherry-GFP-LC3 expression have been described (6). Adenoviral
transcriptionofautophagy-lysosomemachinerygenes(6).Taken delivery of constructs was employed to achieve a high efficiency of
together with the in vivo observation that haploinsufficiency of transductionandpermitevaluationofdose-dependenteffectsinpri-
beclin-1bytargeteddisruptionofaBECN1alleleconferscytopro- maryNRCMs,aspreviouslydescribed(6,15).Adenoviralparticlesfor
expression of short hairpin RNAs (shRNAs) targeting rat TFEB,
tectionincardiacIRinjury(5),thesedatasuggestthehypothesis
BECN1,andPPARGC1(cid:2)weregeneratedwiththeBLOCKiTadenoviral
thatROS-inducedupregulationofbeclin-1transcriptionallyim-
system (Invitrogen). Specific oligonucleotide sequences employed
pairs the lysosomal machinery to prevent removal of damaged
were as follows (with targeted coding sequences underlined): (i)
mitochondria and cause cell death with BNIP3 expression and
shRNAtargetingratTFEB,5=-CACCGAATCAAGGAGCTGGGAATG
hypoxia-reoxygenationinjury.Inthisstudy,weuncoveredanau- CTGATCGAAATCAGCATTCCCAGCTCCTTGATTC-3=(top-strand
toregulatoryloopwherebybeclin-1levelsregulateTFEBactivity, oligonucleotide) and 5=-AAAAGAATCAAGGAGCTGGGAATGC
which coordinates mitochondrial autophagy with biogenesis to TGATTTCGATCAGCATTCCCAGCTCCTTGATTC-3= (bottom-strand
controlmitochondrialqualityandregulatestress-inducedcardi- oligonucleotide);(ii)shRNAtargetingBECN1(annotatedshBECN1-2),
omyocytedeath. 5=-CACCGCTCAGTACCAGCGAGAATATCGAAATATTCTCGCTGG
TACTGAGC-3= (top-strand oligonucleotide) and 5=-AAAAGCT
CAGTACCAGCGAGAATATTTCGATATTCTCGCTGGTACTGA
MATERIALSANDMETHODS
GC-3= (bottom-strand oligonucleotide); and (iii) shRNA targeting rat
Invivoischemia-reperfusionmodeling.BECN1heterozygousnullmice PPARGC1(cid:2), 5=-CACCGAGCAAGTATGACTCTCTGCGAACAGAGAG
(BECN1(cid:3)/(cid:4);micewithhaploinsufficiencyofbeclin-1)(26)andwild-
TCATACTTGCTC-3= (top-strand oligonucleotide) and 5=-
type (WT) mice of the C57BL/6 strain were obtained from Jackson
AAAAGAGCAAGTATGACTCTCTGTTCGCAGAGAGTCAT
Laboratories and subjected to reversible left anterior descending
ACTTGCTC-3=(bottom-strandoligonucleotide).
(LAD)coronaryarteryocclusiontoinduceischemiafor30min,fol-
Adenoviral particles for shRNAs targeting BECN1 (annotated
lowedby4hofreperfusion,asdescribedpreviously(6).Briefly,mice
shBECN1-1) (5) and LacZ, as a nontargeting control for studies with
were anesthetized with a mixture of ketamine (80 mg/kg of body
shRNA-mediatedknockdown,andforoverexpressionofLacZ(asacon-
weight) and xylazine (5 mg/kg), surgically prepped, and ventilated.
trol), green fluorescent protein (GFP)-tagged LC3, FLAG-hBNIP3,
Afterthoracotomy,theLADarterywasidentified,anda9-0polypro-
mBECLIN-1, and hemagglutinin (HA)-tagged hTFEB have been de-
pylenesuturewaspassedundertheLADartery.Aknotwastiedovera
scribed previously (15). Adenoviral particles for expression of FLAG-
1-mmsectionofPE-10tubingplaceddirectlyoverthevesseltocreate
PPARGC1(cid:2)wereengineeredwithataggingcodingsequenceforhuman
theocclusion.Ischemiawasconfirmedbyanabsenceofbloodflow,
PPARGC1(cid:2)withaFLAGtagattheNterminus,usingGatewaycloning
verifiedvisually,andbythepresenceofSTelevationsonelectrocar-
technology(Invitrogen).Viralparticlesweregeneratedperthemanufac-
diogram (EKG). Reperfusion was induced to release the occlusion,
turer’sinstructions.Inallexperiments,controladenoviralparticleswere
whichwasconfirmedbyresolutionofSTsegmentelevation.Atsacri-
added to groups as necessary to ensure equal viral particle doses, i.e.,
fice,theventriclesweresectionedintothreepieces;theapical1/3was
multiplicitiesofinfection(MOIs),betweengroups.
subjectedtobiochemicalanalysisasthe“injured”region,andthebasal
Assessmentofcelldeath.Celldeathassayswereperformedin96-well
1/3wasprocessedasthe“remote”region.Sham-treatedmiceunder-
plates(Nunc,Fisher)withaLive-Deadcytotoxicityviabilitykitformam-
wenttheentiresurgicalprocedurewithoutocclusionoftheLADar-
malian cells (Invitrogen), using a Tecan Infinite M200 Pro microplate
tery. Autophagic flux was assessed in comparison to that of mice
reader(Tecan)followingthemanufacturer’sinstructions,asdescribed
pretreatedwithchloroquine(CQ;40mg/kg,administeredintraperi-
previously(6).
toneally[i.p.])30minpriortoIRmodeling,aspreviouslydescribed(6,
AssessmentofTUNELpositivity.Terminaldeoxynucleotidyltrans-
27).AllanimalstudieswereapprovedbytheAnimalStudiesCommit-
ferase-mediateddUTP-biotinnickendlabeling(TUNEL)wasperformed
teeattheWashingtonUniversitySchoolofMedicineandtheInstitu-
onfixedandpermeabilizedNRCMsbyusingtheDead-Endfluorometric
tionalAnimalCareandUseCommitteeattheJohnCochranVAMed-
TUNEL system (Promega) following the manufacturer’s directions, as
icalCenter.
Cellculture.Isolationofneonatalratcardiacmyocytes(NRCMs) previouslydescribed(15).
wasperformedasdescribedpreviously(6).Heartswereremovedfrom CellularATPcontentanalysis.NRCMsweretransducedwithadeno-
1-day-old Sprague-Dawley rats, the atria and great vessels were viralparticlesfor24handthentrypsinized,and5(cid:7)104cellswereplated
trimmedoff,andtissuewasfinelyminced,followedbysequentialdi- ineachwellofa96-wellplate.CellularATPlevelswerequantifiedusinga
gestionwith0.5mg/mlcollagenase(WorthingtonBiochemical,NJ). luciferasereaction-basedassaykit(CellTiter-Gloreagent;Promega),and
Ventricularcardiomyocyteswereseparatedfromfibroblastsbydiffer- readingswerenormalizedtothoseofviablecellsassessedbythetrypan
entialplatinginmediumcontainingDulbecco’smodifiedEagle’sme- blueexclusionmethodaspreviouslydescribed(28).
dium(DMEM),10%horseserum,5%fetalcalfserum,100(cid:5)mol/liter Immunofluorescenceimaging.ImagingformCherry-GFP-LC3and
bromodeoxyuridine, penicillin, streptomycin, and L-glutamine, fol- LysoTrackerRedwasperformedwithliveNRCMs,andimagingforGFP-
lowedbytissuecultureingelatin-coatedplatesinmediumcontaining LC3,LAMP1,andTFEB(withimmunofluorescenceoftheHAtag)was
modifiedDMEM,100(cid:5)Mbromodeoxyuridine,penicillin,streptomy- performedwithNRCMsfixedwith4%paraformaldehyde(20min)and
cin, L-glutamine, 10 ng/ml insulin, 10 (cid:5)g/ml transferrin, and 0.5 thenpermeabilizedwith0.1%TritonX-100(5min),usinganAxioscap
mg/mlbovineserumalbumin. uprightmicroscopefittedwithanAxioCamHRCcameraandPlanNeo-
Nutrient deprivation was induced by culture in serum-free Hanks fluarobjectives(20(cid:7)[numericalaperture{NA}(cid:8)0.60],40(cid:7)[NA(cid:8)
balancedsaltsolution(HBSS;Invitrogen).HEK293cellswereculturedas 0.75],and63(cid:7)[oil][NA(cid:8)1.25][Zeiss]),alongwithappropriatefilter
describedpreviously(15). cubes,aspreviouslydescribed(6).NuclearlocalizationofTFEBwasas-
Hypoxia modeling. Cells were subjected to hypoxia in vitro in an sessedin60cells/group.LAMP1-positivepunctain50cells/groupwere
oxygen control cabinet (Coy Laboratories, Grass Lake, MI) mounted countedusingImageJsoftwareasdescribedpreviously(15),andresults
withinanincubatorandequippedwithanoxygensensorforcontinuous are expressed as numbers of dots/nucleus. Imaging for BNIP3 and
oxygenlevelmonitoring.Amixtureof95%nitrogenand5%CO was COX-IVwasperformedonfixedandpermeabilizedNRCMswithaZeiss
2
infusedtocreatehypoxia,andoxygenlevelsinthechamberweremoni- confocalLSM700laserscanningconfocalmicroscopeusing63(cid:7)Zeiss
toredandmaintainedat(cid:6)1%,asdescribedpreviously(6). Plan-Neofluar 40(cid:7)/1.3 and 63(cid:7)/1.4 oil immersion objectives. Nuclei
March2015 Volume35 Number6 MolecularandCellularBiology mcb.asm.org 957
Maetal.
werestainedbluewithHoechstdye(forlive-cellimaging)(H3750;In- HEK293cellsweretransfectedwiththePGL3.Lucvector,drivenbythe
vitrogen) or DAPI (4=,6-diamidino-2-phenylindole) (for fixed cells) humanPPARGC1(cid:2)promoterwithout(i.e.,wild-typepromoter)orwith
(H1200;VectorLaboratories).Epifluorescenceandconfocalimageswere mutationsinthe“CLEARsequence,”located69bpupstreamofthetran-
acquired and analyzed using Zeiss Axiovision and Zen 2010 software, scriptionalstartsite(i.e.,(cid:4)18CLEARsitemutant),togetherwithpRL-
respectively. SV40forRenillaluciferaseexpression(ataratioof100:1).Inaddition,
Flow cytometric assessment. NRCMs were incubated with Lyso- cellswerecotransfectedwithexpressionvectorscodingfortheconstitu-
TrackerRed(1mmol/literfor15min)toassesslysosomeabundance,with tivelyactivetranscriptionfactorCREB,theCREBactivatorTORC2(i.e.,
nonyl-acridineorange(NAO)(acardiolipin-bindingdye;10nmol/liter CRTC2)(seebelowfordetails),TFEB(pAd-CMV/V5-hTFEB),orLacZ
for15min)andMitotrackerGreen(200nmol/literfor45min)toassess (pAd-CMV/V5-LacZ)(asacontrol),andluciferaseactivitiesweremea-
mitochondrialmass,withJC-1(10mg/mlfor10min)ortetramethylrho- sured48haftertransfection.Fireflyluciferasereadingswerenormalized
damine,ethylester(TMRE;50nmol/literfor30min)toassessmitochon- tothoseforRenillaluciferasetocontrolfortransfectionefficiency.
drialpolarization,andwithcarboxy-H DCFDA(acell-permeatingROS Subcellular fractionation. NRCMs were subjected to biochemical
2
indicator;10(cid:5)mol/literfor30min)toassessthelevelsofROS.Following fractionationtorecovernucleus-enrichedandcytoplasmicfractions,
incubation with these compounds at 37°C in 5% CO , the cells were aspreviouslydescribed(23).Heartswerefractionatedintonucleus-
2
trypsinizedandsubjectedtoflowcytometryonaFACScaninstrument enrichedandcytoplasmicsamplesbyusingaCelLyticNuCLEARex-
(Becton-Dickinson)aspreviouslydescribed(15).Cyflogicsoftware(Cy- tractionkit(Nxtract;Sigma).Expressionofproteinslocalizedtothe
Flo)wasemployedtoanalyze20,000eventsperrun. nucleus(histoneH3)andcytoplasm(glyceraldehyde-3-phosphatede-
Assessment of mitochondrial DNA content. Mitochondrial DNA hydrogenase [GAPDH]) was examined to confirm relative enrich-
contentwasassessedbyquantitativePCRanalysisaspreviouslydescribed ment.
(29).Briefly,DNAwaspreparedfromNRCMpellets,andreal-timequan- Coimmunoprecipitation studies. Murine embryonic fibroblasts
titativePCRwasperformedforthemitochondrialgeneNADH2(with (MEFs)fromC57BL/6wild-typemicewereadenovirallytransducedwith
primers5=-ATCACCACCATTCTCGCAAT-3=and5=-TCCTATGTGGG beclin-1–HA(MOI(cid:8)100)ortransfectedwithN-terminalHA-tagged
CAATTGATG-3=)andthenucleargeneRCAN1(withprimers5=-GGTT TSC1(Addgene)(32).Threehundredmicrogramsofproteinwasincu-
TGCTGAGCCTCGAAG-3=and5=-CTTCATCCCTCCTTTGTAAC-3=). batedwithanti-HA(H3663;Sigma)ornormalrabbitIgG,andimmuno-
Theratioofcopiesofmitochondrialtonucleargenesrepresentstherela- precipitationwasperformedusingDynaBeads(Invitrogen).
tivemitochondrialDNAcontent. Immunoblotting. Immunoblotting was performed on cellular ex-
Assessmentofmitochondrialfragmentation.Mitochondrialmor- tracts by use of previously described techniques (15). Antibodies em-
phologywasascertainedtobefragmentedortubularbyvisualinspection ployedwereasfollows:antibodiesforFLAG(F3165;Sigma),HA(H6908;
ofindividualcellsatahighmagnification,asdescribedpreviously(30). Sigma),LAMP1(AB24170;Abcam),beclin-1(ab16998[Abcam]andsc-
Fifty cells were evaluated per experimental group, and results are ex- 11427[SantaCruz]),p70S6K(2708;CellSignaling),p-p70S6K(9234;Cell
pressedaspercentagesofcellsshowingpredominantlyfragmentedmito- Signaling),4-EBP1(9644;CellSignaling),p-4EBP1(2855;CellSignaling),
chondria. p-mTOR(2974;CellSignaling),mTOR(2983;CellSignaling),histoneH3
Assessmentofmitochondrialpolarization.Mitochondrialdepolar- (9715;CellSignaling),TFEB(MBS120432;employedtodetectadenovi-
izationwasassessedbyexpressionofTMREorwiththeratiometricdye rallytransducedHA-taggedhumanTFEBwhenHAdetectionwaspre-
JC-1,whichaggregatesintoredfluorescentpolymersinnormallypolar- cludedforspecificity),GAPDH(ab22555;Abcam),peroxisomeprolifera-
izedmitochondriabutispredominantlyobservedasgreenfluorescent tor-activated receptor gamma coactivator 1(cid:2) (PGC1(cid:2)) (ab106814;
monomers in cells with depolarized mitochondria, as previously de- Abcam),tuberoussclerosiscomplex1(TSC1)(6935;CellSignaling),and
scribed(6,15). tuberoussclerosiscomplex2(TSC2)(3612;CellSignaling).MurineTFEB
TEM.NRCMswerefixedinmodifiedKarnovsky’sfixative(3%glu- was detected with a Bethyl Labs antibody (A303-673A). The multiple
taraldehyde,1%paraformaldehydein0.1Msodiumcacodylatebuffer), immunoreactiveTFEBbandsobserved(seeFig.12CandD)likelyrepre-
followedbypostfixationin2%osmiumtetroxidein0.1Msodiumcaco- sent various TFEB isoforms (NP_001155194.1 and NP_035679.3) or
dylatebufferfor1h.Thecellswerethenstainedwith2%aqueousuranyl posttranslationally modified forms of the protein in the myocardium.
acetatefor30min,dehydratedinaseriesofgradedethanolconcentra- Immunoblotting for (cid:2)-sarcomeric actin ((cid:2)SA) (ab52219; Abcam) was
tions,andembeddedinPolyBed(Polysciences).Blocksweresectionedat employedasaloadingcontrolunlessotherwiseindicated.ImageJsoftware
a90-nmthickness,poststainedwithVenable’sleadcitrate,andviewed wasemployedforquantitativeanalysis.Proteinabundancewasnormal-
with a JEOL model 1200EX transmission electron microscope (TEM) izedto(cid:2)-sarcomericactinproteinexpressionandreportedasthefold
(JEOL,Tokyo,Japan).DigitalimageswereacquiredusinganAMTAd- changeversusthecontrol.Chemicalsemployedwereasfollows:3-methyl-
vantageHR(AdvancedMicroscopyTechniques,Danvers,MA)high-def- adenine(3MA;EMD4Biosciences),torin-1(Tocris),MG-132(Cayman
initioncharge-coupleddevice(CCD)1.3-megapixelTEMcamera. ChemicalCompany),andMnTMPyP(Calbiochem).Equivalentquanti-
Assessmentofpromoter-drivenluciferaseactivity.Atotalof3(cid:7)104 ties of appropriate diluent were employed as controls in all studies in
cells were plated per well of a 96-well plate and transfected with the whichthesechemicalswereemployed.
PGL3.Lucvector,carryinga2,760-bpfragmentofthehumanPPARGC1(cid:2) QuantitativePCRanalysisformRNA.Real-timePCRwasperformed
promoter(forfireflyluciferaseexpression),andthepRL-SV40vector(for asdescribedpreviously(6).Briefly,totalRNAwaspreparedfromNRCMs
Renillaluciferaseexpression[31])ata100:1ratio,usingLipofectamine byuseofanRNA-easyminikit(Qiagen),andcDNAwassynthesizedwith
2000 (Invitrogen). Constructs coding for the human PPARGC1(cid:2) pro- 1(cid:5)goftotalRNAbyusingtheSuperScriptIIIfirst-strandsynthesissystem
moter(GenBankaccessionnumberNG_028250.1)withindividualmu- (Invitrogen).OnemicroliterofcDNAtemplatewasmixedwith12.5(cid:5)lof
tationsinthethreeCLEARsitesdetectedwithin(asdepictedinFig.9D) 2(cid:7)SYBRgreenPCRmastermix(Invitrogen)andsubjectedtoquantita-
weregeneratedbysite-directedmutagenesisusingaQuikChangeLight- tivePCRintriplicateunderthefollowingconditions:50°Cfor2minand
ningsite-directedmutagenesiskit(Stratagene)andverifiedbysequenc- 95°Cfor10minfollowedby40cyclesof95°Cfor15sand60°Cfor1min
ing.Luciferaseactivitywasmeasuredwithadual-luciferaseassaykit(Pro- inanABI7500Fastreal-timePCRsystem.TheGAPDHgenewasalso
mega) and a Tecan Infinite M200 Pro microplate reader (Tecan) amplifiedinparallelasareferenceforthequantificationoftranscripts.
followingthemanufacturers’instructions.Fireflyluciferasevalueswere PrimersetsemployedareshowninTable1.Resultswerecalculatedusing
normalizedtothoseforRenillaluciferasetocontrolfortransfectioneffi- the(cid:9)(cid:9)C methodandexpressedaspreviouslydescribed(6).
T
ciency.Toassesswhetherthebp(cid:4)18sitemutantconstructretainedac- ChIP assays. Chromatin immunoprecipitation (ChIP) was per-
tivitytodrivetranscriptionalactivation(otherthanthatdrivenbyTFEB), formedaspreviouslyreported(25,33).Briefly,HEK293Acellsweretrans-
958 mcb.asm.org MolecularandCellularBiology March2015 Volume35 Number6
Beclin-1RegulatesMitochondrialQualityviaTFEB
TABLE1PrimersequencesemployedforqPCRanalysisoftheindicatedgenes
Primersequence(5=–3=)
Targetgene Forward Reverse
Ratgenes
MAP1LC3B TTTGTAAGGGCGGTTCTGAC CAGGTAGCAGGAAGCAGAGG
SQSTM1 GCTGCCCTGTACCCACATCT CGCCTTCATCCGAGAAAC
LAMP1 TCTTCAGCGTGCAAGTCCAG ATGAGGACGATGAGGACCAG
PPARGC1(cid:2) ATGAATGCAGCGGTCTTAGC AACAATGGCAGGGTTTGTTC
BECN1 TTCAAGATCCTGGACCGAGTGAC AGACACCATCCTGGCGAGTTTC
TFEB GTCCAGCAGCCACCTGAACGT ACCAGGGAAGCCGTGACCTG
LAMP2A CCAAATTGGGATCCTAACCTAA TGGTGAAGCAGTGTTTATTAATTCC
LAMP2B GGTGCTGGTCTTTCAGGCTTGATT ACCACCCAATCTAAGAGCAGGACT
CTSB AAATCAGGCGTATACAAGCATGAA AGCCCAGAATGCGGATGG
CTSD CTGAGTGGCTTCATGGGGAT CCTGACAGTGAAGAAGGAGC
RAB7 TTACTTCGAGACCAGTGCCAAGGA TGTCCAGTTTGATGGGTTCAGGGA
GAPDH GGCCGAGGGCCCACTA TGTTGAAGTCACAGGAGACAACCT
RPS12 AAGGCATAGCTGCTGGAGGTGTAA AGTTGGATGCGAGCACACACAGAT
RPL32 CCTCTGGTGAAGCCCAAGATC TCTGGGTTTCCGCCAGTTT
Mousegenes
MAP1LC3B CGTCCTGGACAAGACCAAGT ATTGCTGTCCCGAATGTCTC
LAMP1 TCTTCAGTGTGCAGGTCCAG ATGAGGACGATGAGGACCAG
PPARGC1(cid:2) ATGAATGCAGCGGTCTTAGC AACAATGGCAGGGTTTGTTC
BECN1 AATCTAAGGAGTTGCCGTTATAC CCAGTGTCTTCAATCTTGCC
TFEB GTCTAGCAGCCACCTGAACGT ACCATGGAGGCTGTGACCTG
LAMP2A CCAAATTGGGATCCTAACCTAA TGGTCAAGCAGTGTTTATTAATTCC
LAMP2B GGTGCTGGTCTTTCAGGCTTGATT ACCACCCAATCTAAGAGCAGGACT
RAB7 TTACTTCGAGACCAGTGCCAAGGA TGTCCAGTTTGATGGGTTCAGGGA
CTSB AAATCAGGAGTATACAAGCATGA GCCCAGGGATGCGGATGG
CTSD CTGAGTGGCTTCATGGGAAT CCTGACAGTGGAGAAGGAGC
SQSTM1 GCTGCCCTATACCCACATCT CGCCTTCATCCGAGAAAC
GAPDH ACTCCCACTCTTCCACCTTC TCTTGCTCAGTGTCCTTGC
RPS12 AAGACCGCCCTCATCCACGATG TTGGTGCTCAGCACAAAGTGCC
RPL32 CCTCTGGTGAAGCCCAAGATC TCTGGGTTTCCGCCAGTTT
fectedwiththeemptypAAV-CMV-TFEB-IRES-GFPconstruct,described verified, following which statistical differences were assessed with un-
earlier(34),orwithemptyvector(asacontrol)for48h.Cellswerecol- paired2-tailedStudent’sttestfortwoexperimentalgroups,one-wayanal-
lected,fixedwith1%formaldehydefor10min,andlysedonicewithChIP ysisofvariance(ANOVA)formultiplegroups,andtwo-wayANOVAfor
lysisbuffer(50mMTris-HCl,pH7.5,100mMNaCl,1%TritonX-100, testingoftwovariablesacrossmultiplegroups,usingSPSSsoftware.Bon-
1%Tween20)for20min.Thelysatewasdigestedwithmicrococcalnu- ferroni’sandDunnett’sT3posthoctestswereemployedafterANOVAfor
clease(Sigma)for15minat37°C,andthereactionwasterminatedusing testingforsignificantdifferencesbetweengroupsfordatawithequaland
EDTA(2mM)andsodiumdodecylsulfate(SDS;1%).SDS-Out(Pierce) unequalvariances,respectively.Nonparametrictestswereemployedfor
wasemployedtoprecipitatetheunboundSDS,andlysatesdiluted1:1 datathatwerenotnormallydistributed.Atwo-tailedPvalueof(cid:6)0.05was
withChIPdilutionbuffer(50mMTris-HCl,pH7.5,100mMNaCl,0.5% consideredstatisticallysignificant.
TritonX-100,2mMEDTA)wereincubatedwithhigh-capacityNeutr-
Avidinagarose(Pierce).Biotinylatedanti-FLAGantibody(F9291;Sigma) RESULTS
or isotype control IgG1 (M9035; Sigma) was precoupled with Neutr-
BNIP3expressionincreasesbeclin-1abundancewithtranscrip-
Avidinbeadsfor30minat4°C,andtheprotein-DNAcomplexeswere
tionalsuppressionofautophagy-lysosomemachinery.BNIP3,a
immunoprecipitated overnight at 4°C. After six washes, the DNA was
elutedbyadditionof8mMbiotin,1%SDSinTris-EDTA(TE)bufferand Bcl-2familyprodeathprotein,istranscriptionallyinducedincar-
precipitatedafterreversingthecross-linkagebyuseof200mMNaClat diacIRinjuryandprovokesmitochondrialpermeabilization(8,
65°Covernight.PCRwasperformedusingprimersflankingtheCLEAR 11,12).WehaveobservedthatBNIP3expressionprovokespro-
site18bpupstreamofthetranscriptionalstartsite(forwardprimer,5=-C gressivedeclinesinlysosomenumbers,withaccumulationoffrag-
GTCACGAGTTAGAGCAGCA-3=;andreverseprimer,5=-TCGCCCTTA mentedanddepolarizedmitochondria,leadingtocelldeath(15),
AGCTTTTTCAA-3=)(204-bpamplicon).Anadditionalprimersetwithin and that restoring lysosome biogenesis by exogenous TFEB ex-
an adjacent segment located between the CLEAR sites at bp (cid:4)18 and
pressionreestablishesmitochondrialautophagytorescueBNIP3-
(cid:4)469 (forward primer, 5=-CTGTCATGAAACAGGGAGCTTT-3=; and
inducedcelldeath(15).Toexaminethemechanismsforimpaired
reverseprimer,5=-GCTTTGAATGCCACAGACTCTA-3=)(125-bpam-
autophagicremovalofBNIP3-damagedmitochondria,weevalu-
plicon)didnotgenerateaproduct,demonstratingspecificity(datanot
atedtheregulationoftheautophagy-lysosomemachinerybyad-
shown).
Statisticalanalysis.Resultsareexpressedasmeans(cid:10)standarderrors enoviraltransductionofNRCMswithBNIP3.Wehypothesized
ofthemeans(SEM),withthesamplesize(n)pergroupindicatedinthe thatthedeclineinlysosomenumberisdrivenbyBNIP3-induced
figurelegends.Assumptionsofnormalityandequalityofvariancewere autophagy,asalsoobservedearlyduringstarvation-inducedau-
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FIG 1BNIP3 expression results in transcriptional downregulation of autophagy-lysosome machinery. (A) Confocal images demonstrating LysoTracker
fluorescence(top)andLAMP1expression(bottom)inNRCMstransducedwithBNIP3(green)orLacZ(24h),withorwithout3MA(7mmol/liter)andwith
quantitation of the number of LAMP1-positive dots/nucleus. *, #, and $, P (cid:6) 0.05 versus LacZ, BNIP3, and BNIP3 plus 3MA, respectively. (B and C)
Representativeflowcytometrictracings(B)andquantitation(C)ofLysoTrackerfluorescence(n(cid:8)3).*,P(cid:6)0.05versuscontrol(con)(whitebar).(DandE)
Representativeimmunoblot(D)andquantitation(E)forLAMP1inNRCMstreatedasdescribedforpanelA(n(cid:8)3).*,P(cid:6)0.05versuscontrol(whitebar).(F
toH)Representativeimmunoblot(aspreviouslydescribed[15])(F)andquantitationofLAMP1protein(n(cid:8)3)(G)andLAMP1transcripts(n(cid:8)4to6)(H)in
NRCMs transduced with BNIP3 or LacZ for the indicated durations. (I) Levels of representative autophagy-lysosome machinery transcripts in NRCMs
transducedwithBNIP3orLacZ(24h;n(cid:8)8).AlltransductionswereperformedatanMOIof100/sample.
tophagy (18). However, BNIP3-induced declines in lysosome anism for induction of autophagosome formation (14), we
abundance (assessed with the acidophilic dye LysoTracker Red examinedtheeffectofBNIP3expressiononbeclin-1.Beclin-1
[Fig.1AtoC]andbyimmunostainingforLAMP1[acandidate protein abundance was increased early (at 12 and 24 h) after
lysosomalproteinwhoselevelsareusedtotracklysosomeabun- BNIP3 transduction (Fig. 2A and B), and this was not due to
dance{35}][Fig.1A])andinLAMP1proteinabundance(Fig.1D transcriptionalinduction(Fig.2C).Infact,BECN1transcript
andE)wereonlypartiallyrestoredwith3MA(atypeIIIphospha- levelsweresignificantlyreducedcomparedwiththoseofcon-
tidylinositol 3-kinase inhibitor which inhibits autophagosome trols at 24 h, coinciding with the suppression of other au-
formation)(Fig.1AtoE),to65%,59%,and72%,respectively,of tophagy-lysosome machinery transcripts (Fig. 1I), and only
thelevelsforsimilarlytreatedcontrols.Thisindicatesthatlyso- increased subsequently, in parallel with a decline in beclin-1
some consumption in BNIP3-induced autophagy is only partly
proteinabundance(Fig.2BandC)secondarytoconsumption
responsiblefortheobserveddeclineinlysosomeabundance(15).
withpersistentautophagy(datanotshown).Thissuggeststhat
Importantly, we observed progressive declines in LAMP1 tran-
a stabilization of beclin-1 protein results in its accumulation
scripts(Fig.1H)parallelingthedeclineinLAMP1proteinabun-
earlyafterBNIP3expression.
dance(Fig.1FandG).Thistranscriptionalsuppressionalsoaf-
We next examined if ROS mediate the BNIP3-induced in-
fected multiple proteins (Fig. 1I) that participate in autophagy
creaseinbeclin-1abundanceobservedwithreperfusioninjury
(LC3, p62, and RAB7), constitute the lysosomes (LAMP2A,
(6).BNIP3triggeredROSgenerationearlyafteritsexpression
LAMP2B,cathepsinB,andcathepsinD),andfunctionasthemas-
(at 12 h) (Fig. 2D and E), likely as a result of mitochondrial
tertranscriptionalregulatoroftheautophagy-lysosomemachin-
depolarization (Fig. 2F and G). Scavenging of ROS with
ery(TFEB)butdidnotalterexpressionoftheunrelatedribosomal
MnTMPyP,acell-permeatingsuperoxidedismutasemimetic,
proteinsRPL32andRPS12(changesintranscript/GAPDHabun-
dancesof0.95-(cid:10)0.02-fold[versus1.01-(cid:10)0.03-foldforLacZ] did not affect BNIP3-induced mitochondrial depolarization
and0.95-(cid:10)0.03-fold[versus1.04-(cid:10)0.03-foldforLacZ],respec- (Fig. 2F and G) but prevented the increase in beclin-1 abun-
tively; the P value was nonsignificant [NS]; n (cid:8) 8). Taken to- dance (Fig. 2H and I), indicating that BNIP3-induced mito-
chondrialpermeabilizationisthecauseratherthantheconse-
gether, these observations indicate that in contrast to the well-
quenceofROSgeneration,whichprovokesincreasedbeclin-1
describedtranscriptionalstimulationoftheautophagy-lysosome
machinery observed with starvation (21–24), BNIP3 expression abundance.TreatmentwithMnTMPyPinducedreductionsin
resultsintranscriptionalsuppressionofthismachinerytoprevent LAMP1transcripts(Fig.2J)andproteinlevels(Fig.2KandL),
itsreplenishment,whileautophagyisrobustlyinducedtoremove consistent with prior reports indicating that basal ROS levels
BNIP3-damagedmitochondria(15). maydrivetranscriptionoflysosomalgenes(36).Importantly,
We and others have observed a rapid increase in beclin-1 treatmentwithMnTMPyPpreventedBNIP3-induceddeclines
abundance with reperfusion/reoxygenation injury (5, 6), and inLAMP1transcripts(Fig.2J)andprotein(Fig.2KandL)and
elevated beclin-1 levels transcriptionally suppress autophagy- inlysosomes(Fig.2MandN)andattenuatedBNIP3-induced
lysosomemachinerygenes(6).SinceBNIP3expressionispos- cardiomyocytedeath(Fig.2O).Takentogether,theseobserva-
tulatedtodisplacebeclin-1fromitsbindingtoBcl2asamech- tions indicate that BNIP3-induced ROS mediate increased
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Beclin-1RegulatesMitochondrialQualityviaTFEB
FIG2BNIP3-inducedROSdriveincreasedbeclin-1proteinlevelsandsuppressionoflysosomeabundance.(AtoC)Representativeimmunoblot(A)and
quantitationofbeclin-1protein(n(cid:8)3)(B)andBECN1transcripts(n(cid:8)3or4)(C)inNRCMstransducedwithBNIP3orLacZfortheindicateddurations.(D
andE)Representativeflowcytometrictracings(D)andquantitation(E)ofROS(n(cid:8)3or4)inNRCMstransducedwithBNIP3orLacZfor12h.HO (500
2 2
mmol/liter;4h)wasusedasthepositivecontrol.(FandG)Representativeflowcytometrictracings(F)andquantitation(G)ofTMREfluorescence(n(cid:8)3)in
NRCMstreatedasdescribedforpanelD.(HandI)Representativeimmunoblot(H)andquantitation(I)ofbeclin-1(n(cid:8)3or4)inNRCMstransducedwith
BNIP3orLacZplusMnTMPyP(50(cid:5)mol/liter;24h).(JtoL)LAMP1transcript(n(cid:8)6)(J)andprotein(representativeimmunoblot[K]andquantitation[L]
[n(cid:8)4])abundancesinNRCMstreatedasdescribedforpanelH.(MandN)Representativeflowcytometrictracings(M)andquantitation(N)ofLysoTracker
fluorescence(n(cid:8)3).(O)CelldeathinNRCMstreatedasdescribedforpanelH(n(cid:8)7or8).AlltransductionswereperformedatanMOIof100/sample.*,P(cid:6)
0.05versuscontrol(whitebars).
beclin-1abundanceandtheobservedtranscriptionalsuppres- therelativeabundanceofautolysosomesandreducedthelevelof
sionofthelysosomemachinery. autophagosomes (versus the shLacZ control) in BNIP3-trans-
Partialbeclin-1knockdowntranscriptionallystimulatesau- ducedNRCMs(Fig.3GandH),pointingtoenhancedautophagic
tophagic flux to attenuate BNIP3-induced cardiomyocyte flux. Partial beclin-1 knockdown also resulted in an increased
death. We have observed that experimental modulation of abundanceofautophagyproteins(totalLC3andp62)(Fig.3I),
beclin-1levelstranscriptionallyaffectsautophagy-lysosomema- which was transcriptional as we described earlier (6; data not
chinery proteins in a reciprocal fashion (6). To determine if shown),andinLAMP1(Fig.3IandJ),parallelingtheincreased
BNIP3-induced upregulation of beclin-1 contributes to the ob- lysosome abundance. Importantly, partial beclin-1 knockdown
serveddeclineinLAMP1,weperformedapartialknockdownof attenuatedtheBNIP3-induceddeclineinLAMP1protein(Fig.3I
BECN1 transcripts (with two different shRNA constructs) in andJ),reducedBNIP3-inducedTUNELpositivity(Fig.3KandL),
BNIP3(andLacZ)-treatedNRCMs.Partialbeclin-1knockdown and prevented BNIP3-induced cell death (Fig. 3M), mimicking
(reductioninBECN1transcriptsto(cid:11)69%ofcontrollevelandin the observations seen with exogenous expression of TFEB (15).
beclin-1proteinto(cid:11)45to50%ofcontrollevel)(Fig.3AtoC) Interestingly, partial beclin-1 knockdown resulted in reduced
stimulated LAMP1 transcription (Fig. 3D) and increased lyso- BNIP3protein,whichwaspreventedbyconcomitantinhibition
someabundance(Fig.3EandF)topreventBNIP3-inducedde- ofautophagosomeformationwith3MA(Fig.3N)butnotbypro-
clines in lysosome numbers (Fig. 3E and F). BNIP3 expression teasome inhibition with MG-132, suggesting an enhanced au-
resulted in autophagosome accumulation, indicating impaired tophagicdegradationofBNIP3togetherwiththemitochondria,
autophagicflux(15),andpartialbeclin-1knockdownincreased whereupon it is localized. Partial beclin-1 knockdown with the
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FIG4Nearlycompletebeclin-1knockdowninhibitsautophagytoincreaseBNIP3-inducedcelldeath.(A)BECN1transcriptlevelsinNRCMstransducedwith
shBECN1-1,shBECN1-2,orshLacZ(MOI(cid:8)10;72h).(B)Representativeimmunoblot(top)andquantitation(bottom)ofbeclin-1proteinabundancein
NRCMstreatedasdescribedforpanelA(n(cid:8)4).*,P(cid:6)0.05versusshLacZ.(CandD)GFP-taggedLC3localization(magnification,(cid:7)630;representativeof3
experiments)(C)andquantitationofpunctateGFP(D)inNRCMstreatedasdescribedforpanelAandcotransducedwithBNIP3orLacZ(24h)(n(cid:8)15to20
cells/group).*and#,P(cid:6)0.05versuscontrol(whitebars)andtheBNIP3-plus-shLacZgroup,respectively.(E)CelldeathinNRCMstreatedasdescribedforpanel
C(n(cid:8)7to15).*,P(cid:6)0.05versuscontrol(whitebar).
secondshRNAconstruct(shBECN1-2)(Fig.3AtoD)recapitu- Partial beclin-1 knockdown activates TFEB to attenuate
latedthesefindings(datanotshown).Takentogether,thesedata BNIP3-inducedcardiomyocytedeath.Intriguingly,theeffectsof
indicatethatpartialbeclin-1knockdowntranscriptionallystimu- partial beclin-1 knockdown (Fig. 3) mimicked our observa-
latesautophagicfluxtorestoreimpairedautophagosomeprocess- tions with TFEB activation (15). Therefore, we evaluated the
ingwithBNIP3expression. effect of partial beclin-1 knockdown on TFEB activation. Re-
Notably, as we observed previously (6), a more complete centstudiesuncoveredatightlyregulatedmechanismforTFEB
knockdownofBECN1(with10-foldhigheradenoviralshBECN1 activation, whereby TFEB is phosphorylated and sequestered
transduction,to(cid:11)13%oftranscriptlevelswithshLacZ,resulting in the cytoplasm, in the unstressed state, and starvation-in-
inareductionofbeclin-1proteinabundanceto(cid:11)7%ofthecon- duced mTOR inactivation provokes dephosphorylation and
trollevel)(Fig.4AandB)resultedininhibitionofautophagosome nuclear translocation of TFEB (21, 22, 24). Given the lack of
formation (Fig. 4C and D) and increased BNIP3-induced cell suitableantibodiestodetectendogenousTFEBinratcells(24),
death (Fig. 4E), consistent with an obligate role for beclin-1 in weexaminedtheeffectofstarvationonsubcellulardistribution
initiationofautophagy(26). ofexogenousTFEBinNRCMs.ExogenousTFEBexpressedat
FIG3Partialbeclin-1knockdownstimulateslysosomebiogenesisandinducesautophagicfluxtoattenuateBNIP3-inducedcelldeath.(A)BECN1transcript
levelsinNRCMstransducedwithtwoshBECN1constructsorshLacZ(MOI(cid:8)1;72h).(BandC)Representativeimmunoblot(B)andquantitation(C)of
beclin-1protein(n(cid:8)4)inNRCMstreatedasdescribedforpanelA.*,P(cid:6)0.05versusshLacZ.(D)LAMP1transcriptlevelsinNRCMstreatedasdescribed
forpanelAandcotransducedwithBNIP3orLacZ(MOI(cid:8)100;24h)(n(cid:8)4to12).*and#,P(cid:6)0.05versuscontrol(whitebar)andtherespective
shBECN1-treatedgroup,respectively.(EandF)Representativeflowcytometrictracings(E)andquantitation(F)ofLysoTrackerfluorescenceinNRCMs
transducedwithshBECN1-1orshLacZandcotransducedwithBNIP3orLacZasdescribedforpanelD(n(cid:8)3).(GandH)mCherry-GFP-LC3localization
(magnification,(cid:7)630;representativeof3experiments)(G)andquantitationofautophagosomes,autolysosomes,andboth(H)inNRCMstreatedasdescribed
forpanelB(n(cid:8)20to40nuclei/group).*,#,and$,P(cid:6)0.05forautophagosomes,autolysosomes,andboth,respectively,versusshLacZ-plus-LacZcontrol.(Iand
J)Immunoblotdepictingautophagy-lysosomeproteins(I)andquantitationofLAMP1(n(cid:8)3)(J)inNRCMstreatedasdescribedforpanelE.*,P(cid:6)0.05versus
control(whitebar).(KandL)Representativeimages(magnification,(cid:7)200)(K)andquantitationofTUNELpositivity(greeninpanelK)(n(cid:8)4)(L).(M)Cell
deathinNRCMstreatedasdescribedforpanelE(n(cid:8)20to24).*,P(cid:6)0.05versuscontrol(whitebar).(N)BNIP3expressioninNRCMstransducedwith
shBECN1-1orshLacZ(MOI(cid:8)1;72h)andcotransducedwithBNIP3orLacZ(MOI(cid:8)100;24h),withorwithout3MA(7mmol/liter)orMG-132(10
(cid:5)mol/liter).
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