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xld102
2010-06-14 0人阅读 举报 0 0 暂无简介

简介:本文档为《文章pdf》,可适用于工程科技领域

SupportinginformationMankourietalpnasAMPATPAMPKKMOLBKSTRADAICARMetforminAKTHCVβαActiveAMPKAβαSerInactiveAMPKβαPThrEnergyγAMPKPThrγγPdephosphorylationEnergyrequiringprocessesEnergyproducingpathwaysADPATPACCATPADPACCFattyacidcholesterolbiosynthesisPFattyacidcholesterolbiosynthesisHCVreplicationPFigSSchematicoftheregulationofAMPactivatedproteinkinase(AMPK)activityWhenactivatedbyATPconsumptionorpharmacologicalactivation,AMPKisphosphorylatedatthreonineontheαsubunitbyanupstreamcomplex,AMPKkinaseActiveAMPKthenphosphorylatesanumberofsubstratesKeytargetsincludethetwoisoformsofacetylCoAcarboxylase(ACC)Importantly,phosphorylationofACConserinebyAMPKinhibitsenzymaticactivity,concomitantlydecreasingcellularfattyacidsynthesiswhichcouldbedeleterioustohepatitisCvirus(HCV)infectionHCV(mostlikelyviaproteinsNSBandNSA)activatestheserinethreoninekinase,AKT,whichphosphorylatesAMPKonaninhibitoryresidue(serine),leadingtoactivationofACCandincreasedcellularlipidsynthesisMankourietalwwwpnasorgcgicontentshortofSEC)ASGRLucJFHalisedLuciferase(RLUS******NormaControlAICARAMetforminAKTVIII**BSGRLucJFHEC)lisedLuciferase(RLUSMetforminmMAICARmMAµMNormaDCNSABODIPYMergeSGRLucJFHVIIIControldabsorbance(nm)MTTassay(Huh)SAKTVNSABODIPYMergeNormalisedA(µM)AICAR(mM)Metformin(!mM)ControlFigSRestorationofAMPKactivityusingeitherAMPKagonistsorAKTVIIIisdetrimentaltothereplicationofagenotypea(JFH)subgenomicreplicon(SGR)(A)Huhcellsweretransfectedwithinvitrotranscriptsofatransientluciferasesubgenomicreplicon(JFH:genotypea)()andleftuntreated(Control)ortreatedwiththeindicatedcompoundsovernightLuciferaseactivitymeasuredhposttransfectionshowstherelativelevelofgenomereplicationErrorbarsindicatemean±SEM(B)Huhcellsweretransfectedwithinvitrotranscriptsofatransientluciferasesubgenomicreplicon(JFH:genotypea)andtreatedwiththeAMPKagonistsAICAR,A,ormetforminattheindicatedconcentrationsovernightLuciferaseactivitywasmeasuredhposttransfectionandshowstherelativelevelofgenomereplicationErrorbarsshowSEM(C)Huhcellswereleftuntreated(Control)ortreatedwithAICAR,A,ormetforminattheindicatedconcentrationsforhbeforeanalysisofcellviabilityby(,dimethylthiazolyl),diphenyltetrazoliumbromide(MTT)assayErrorbarsindicatemean±SEM(D)DistributionandabundanceofNSAorcellularlipids(BODIPY)wasevaluatedinHuhcellstransfectedwithinvitrotranscriptsofatransientluciferasesubgenomicreplicon(JFH:genotypea)afterfixationandpermeabilizationCellswerestainedforlipidcontentwithBODIPYdyeforhafterNSAlabelingIdenticalsettingsweremaintainedforimagecapturingRepresentativeconfocalimagesareshownCellswereleftuntreated(Control)ortreatedwithAKTVIIIforhbeforeprocessingReplicontransfectedcellsareoutlinedinwhite(Scalebars,μm)TargettAdamsP,McLauchlanJ()DevelopmentandcharacterizationofatransientreplicationassayforthegenotypeahepatitisCvirussubgenomicrepliconJGenVirol:–MankourietalwwwpnasorgcgicontentshortofAntimyc(AMPK)AntiNSAMergeAWildtypeBSDCSAFigSExpressionofanexogenousAMPKSerAlamutantblocksHCVRNAreplicationHuhcellsstablyharboringanHCVgenotypebcultureadaptedsubgenomicreplicon()weretransfectedwiththeindicatedMyctaggedAMPKαconstructsandanalyzedbyimmunofluorescencehposttransfectionRepresentativeconfocalimagesareshownTheupperrowofimagesinA–Cshowsawidefieldimage(Scalebars,μm)KriegerN,LohmannV,BartenschlagerR()EnhancementofhepatitisCvirusRNAreplicationbycellcultureadaptivemutationsJVirol:–MankourietalwwwpnasorgcgicontentshortofAJFHBNSABODIPYMergeReleasedvirusIntracellularvirusinfectivityparedtocontrol)infectivityaredtocontrol)(comp(compControlAICARAMetforminControlAICARAMetforminFigSRestorationofAMPKactivitywithAMPKagonistsinhibitsassemblyandreleaseofinfectiousgenotypea(JFH)virus(A)AdditionalimagesofHuhcellstransfectedwithfulllengthinvitrotranscriptsofJFHCellswerestainedforlipidcontentwithBODIPYdyeforhafterNSAlabelingIdenticalsettingsweremaintainedforimagecaptureRepresentativeconfocalimagesareshownJFHtransfectedcellsareoutlinedinwhiteuntransfectedcellsinthesamefieldareoutlinedinred(Scalebars,μm)(B)HuhcellswereinfectedwithJFHvirusatamultiplicityofinfectionoffocusformingunitspercellAthposttransfection,theindicatedAMPKagonistswereaddedVirusinbothsupernatantandclarifiedcelllysateswastiteredafterafurtherhincubation(n=)Allerrorbarsindicatemean±SEMMankourietalwwwpnasorgcgicontentshortof

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