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Methods in Molecular Biology 1896

Cellular Senescence

Methods and Protocols

M ETHODSIN M OLECULAR B IOLOGY

SchoolofLifeandMedicalSciences

UniversityofHertfordshire Hatfield,HertfordshireAL109AB,UK

Forfurthervolumes: http://www.springer.com/series/7651

CellularSenescence

MethodsandProtocols

MarcoDemaria

ERIBA,UniversityMedicalCenterGroningen,Groningen,Groningen,TheNetherlands

Editor

UniversityMedicalCenterGroningen

Groningen,Groningen,TheNetherlands

ISSN1064-3745ISSN1940-6029(electronic)

ISBN978-1-4939-8930-0ISBN978-1-4939-8931-7(eBook) https://doi.org/10.1007/978-1-4939-8931-7

LibraryofCongressControlNumber:2018962411

© SpringerScience+BusinessMedia,LLC,partofSpringerNature2019

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Preface

Cellularsenescenceisastateofirreversiblegrowtharrest.Originallydescribedasthe mechanismmediatingthelimitedreplicativelifespanofsomaticcells,cellularsenescenceis nowrecognizedasapotenttumor-suppressivemechanism.Moreover,inrecentyears advancementsinthephenotypicalcharacterizationofsenescentcellsunraveledadditional functionalroles,fromdevelopmenttoaging.Thisbookaimsatdescribingcurrentmethods fortheidentificationandcharacterizationofthemajorhallmarksofsenescentcells.Astrong focusreliesonthehighheterogeneityofthesenescence-associatedphenotypes,andtechniquestoinduceandidentifyspecificsenescenceprograms.Moreover,itdescribescellular andmousemodelsinwhichitispossibletostudythecomplexcellandnon-cellautonomous functionsofsenescentcells.

Groningen,Groningen,NLMarcoDemaria

1DetectingCellularSenescenceinReprogramming... .......................1 CoralieCazin,MathieuvonJoest,andHanLi

2DNADamageInSituLigationFollowedbyProximityLigation Assay(DI-PLA) .........................................................11

AlessandroGalbiatiandFabriziod’AddadiFagagna

3ReactiveOxygenSpeciesDetectioninSenescentCells .......................21 StellaVictorelliandJoa ˜ oF.Passos

4CellularIdentificationandQuantificationofSenescence-Associated β-GalactosidaseActivityInVivo ..........................................31 BennettG.Childs,TylerJ.Bussian,andDarrenJ.Baker

5RelativeHumanTelomereLengthQuantificationbyReal-TimePCR. ........39 A.Vasilishina,A.Kropotov,I.Spivak,andA.Bernadotte

6AssessingFunctionalRolesoftheSenescence-AssociatedSecretory Phenotype(SASP).. ....................................................45 NicolasMalaquin,Ve´roniqueTu,andFrancisRodier

7MeasuringtheInflammasomeinOncogene-InducedSenescence .............57 IreneFerna ´ ndez-Duran,Nu ´ riaTarrats,PriyaHari, andJuanCarlosAcosta

8AlarminDetectioninSenescentCells. .....................................71 DongEunKimandAlbertR.Davalos

9IMR90ER:RAS:ACellModelofOncogene-InducedSenescence ............83 AndrewJ.InnesandJesu ´ sGil

10GenotoxicStress-InducedSenescence .....................................93 DorothyN.Y.FanandClemensA.Schmitt

11AMultiparametricAssaytoEvaluateSenescentCells. .......................107 HilahGal,ZivPorat,andValeryKrizhanovsky

12ANovelQuantitativeMethodfortheDetectionofLipofuscin, theMainBy-ProductofCellularSenescence,inFluids ......................119 SophiaV.Rizou,KonstantinosEvangelou,VassiliosMyrianthopoulos, IordanisMourouzis,SophiaHavaki,AikateriniAthanasiou, PanagiotisV.S.Vasileiou,AggelosMargetis,AthanassiosKotsinas, NikolaosG.Kastrinakis,PetrosSfikakis,PaulTownsend, EmmanuelMikros,ConstantinosPantos,andVassilisG.Gorgoulis 13MeasurementofMetaboliteChangesinSenescentCells byMassSpectrometry ...................................................139 ChristopherD.Wiley,SonnetDavis,andArvindRamanathan

14QuantificationofAutophagyDuringSenescence ...........................149 JoonTaePark,Young-SamLee,andSangChulPark

15QuantificationofAneuploidyinMammalianSystems. .......................159 HildavandenBos,BjornBakker,AaronTaudt,VictorGuryev, MariaColome´-Tatche´,PeterM.Lansdorp,FlorisFoijer, andDianaC.J.Spierings

16IdentificationofGenomicAlterationsThroughMultilevel DNAStructuralAnalysis .................................................191 RyanK.ShultzabergerandJohnDresios

17MouseModelsofAcceleratedCellularSenescence... .......................203 MatthewJ.Yousefzadeh,KendraI.Melos,LuiseAngelini, ChristinE.Burd,PaulD.Robbins,andLauraJ.Niedernhofer

18MethodstoQuantifytheNF-κBPathwayDuringSenescence. ...............231 LeiZhang,JingZhao,AditiGurkar,LauraJ.Niedernhofer, andPaulD.Robbins

Index... ...................................................................251

Contributors

JUAN CARLOS ACOSTA EdinburghCancerResearchUKCentre,InstituteofGeneticsand MolecularMedicine,UniversityofEdinburgh,Edinburgh,UK

LUISE ANGELINI InstituteontheBiologyofAgingandMetabolism,UniversityofMinnesota, Minneapolis,MN,USA;DepartmentofBiochemistry,MolecularBiologyandBiophysics, UniversityofMinnesota,Minneapolis,MN,USA;DepartmentofMolecularMedicine,The ScrippsResearchInstitute,Jupiter,FL,USA

AIKATERINI ATHANASIOU DepartmentofObstetricsandGynecology,WeillCornellMedicine, NewYork,NY,USA

DARREN J.BAKER DepartmentofBiochemistryandMolecularBiology,MayoClinic, Rochester,MN,USA;DepartmentofPediatricandAdolescentMedicine,MayoClinic, Rochester,MN,USA

BJORN BAKKER EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA),University ofGroningen,UniversityMedicalCenterGroningen,Groningen,TheNetherlands

A.BERNADOTTE FacultyofMechanicsandMathematics,LomonosovMoscowState University,Moscow,RussianFederation;FederalResearchandClinicalCenterof Physical-ChemicalMedicineofFederalMedicalBiologicalAgency,LaboratoryofSimple Systems,Moscow,RussianFederation

CHRISTIN E.BURD DepartmentofMolecularGenetics,TheOhioStateUniversity, Columbus,OH,USA;DepartmentofCancerBiologyandGenetics,TheOhioState University,Columbus,OH,USA

TYLER J.BUSSIAN DepartmentofBiochemistryandMolecularBiology,MayoClinic, Rochester,MN,USA

CORALIE CAZIN CellularPlasticityandDiseaseModeling,DepartmentofDevelopmental andStemCellBiology,CNRSUMR3738,InstitutPasteur,Paris,France

BENNETT G.CHILDS DepartmentofBiochemistryandMolecularBiology,MayoClinic, Rochester,MN,USA

MARIA COLOME ´ -TATCHE ´ EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA), UniversityofGroningen,UniversityMedicalCenterGroningen,Groningen,The Netherlands;InstituteforComputationalBiology,HelmholtzZentrumMu¨nchen, Neuherberg,Germany

ALBERT R.DAVALOS BuckInstituteforResearchonAging,Novato,CA,USA

SONNET DAVIS BuckInstituteforResearchonAging,Novato,CA,USA

FABRIZIOD’ADDADI FAGAGNA IFOM-Foundation,TheFIRCInstituteofMolecular OncologyFoundation,Milan,Italy;IstitutodiGeneticaMolecolare,ConsiglioNazionale delleRicerche,Pavia,Italy

JOHN DRESIOS LeidosInc.,SanDiego,CA,USA

KONSTANTINOS EVANGELOU MolecularCarcinogenesisGroup,DepartmentofHistologyand Embryology,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens, Greece;DepartmentofAnatomy-Histology-Embryology,MedicalSchool,Universityof Ioannina,Ioannina,Greece

DOROTHY N.Y.FAN DepartmentofHematology,OncologyandTumorImmunology, MolekularesKrebsforschungszentrum–MKFZ,Charite´–UniversityMedicalCenter,Berlin, Germany;GermanCancerResearchCenter(DeutschesKrebsforschungszentrum[DKFZ]),

Heidelberg,Germany;DeutschesKonsortiumfu¨rTranslationaleKrebsforschung(German CancerConsortium),PartnerSiteBerlin,Berlin,Germany

IRENE FERNA ´ NDEZ-DURAN EdinburghCancerResearchUKCentre,InstituteofGenetics andMolecularMedicine,UniversityofEdinburgh,Edinburgh,UK

FLORIS FOIJER EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA),University ofGroningen,UniversityMedicalCenterGroningen,Groningen,TheNetherlands

HILAH GAL DepartmentofMolecularCellBiology,WeizmannInstituteofScience,Rehovot, Israel

ALESSANDRO GALBIATI OncologyIMED,AstraZenecaUKLtd,Cambridge,UK

JESU ´ S GIL MRCLondonInstituteofMedicalSciences(LMS),London,UK;Facultyof Medicine,InstituteofClinicalSciences(ICS),ImperialCollegeLondon,London,UK

VASSILIS G.GORGOULIS MolecularCarcinogenesisGroup,DepartmentofHistologyand Embryology,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens, Greece;FacultyInstituteforCancerSciences,ManchesterAcademicHealthSciencesCentre, UniversityofManchester,Manchester,UK;BiomedicalResearchFoundation,Academyof Athens,Athens,Greece;CenterforNewBiotechnologiesandPrecisionMedicine,Medical School,NationalandKapodistrianUniversityofAthens,Athens,Greece

ADITI GURKAR DepartmentofMolecularMedicineandCenteronAging,TheScripps ResearchInstitute,Jupiter,FL,USA;AgingInstitute,DivisionofGeriatricMedicine, DepartmentofMedicine,UniversityofPittsburgh,Pittsburgh,PA,USA

VICTOR GURYEV EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA), UniversityofGroningen,UniversityMedicalCenterGroningen,Groningen,The Netherlands

PRIYA HARI EdinburghCancerResearchUKCentre,InstituteofGeneticsandMolecular Medicine,UniversityofEdinburgh,Edinburgh,UK

SOPHIA HAVAKI MolecularCarcinogenesisGroup,DepartmentofHistologyandEmbryology, MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens,Greece

ANDREW J.INNES MRCLondonInstituteofMedicalSciences(LMS),London,UK;Faculty ofMedicine,InstituteofClinicalSciences(ICS),ImperialCollegeLondon,London,UK; FacultyofMedicine,CentreforHaematology,ImperialCollegeLondon,London,UK

NIKOLAOS G.KASTRINAKIS MolecularCarcinogenesisGroup,DepartmentofHistologyand Embryology,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens, Greece

DONG EUN KIM BuckInstituteforResearchonAging,Novato,CA,USA

ATHANASSIOS KOTSINAS MolecularCarcinogenesisGroup,DepartmentofHistologyand Embryology,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens, Greece

VALERY KRIZHANOVSKY DepartmentofMolecularCellBiology,WeizmannInstituteof Science,Rehovot,Israel

A.KROPOTOV InstituteofCytology,RussianAcademyofSciences,Saint-Petersburg,Russian Federation

PETER M.LANSDORP EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA), UniversityofGroningen,UniversityMedicalCenterGroningen,Groningen,The Netherlands;TerryFoxLaboratory,BCCancerAgency,Vancouver,BC,Canada; DepartmentofMedicalGenetics,UniversityofBritishColumbia,Vancouver,BC,Canada

YOUNG-SAM LEE WellAgingResearchCenter,DGIST,Daegu,SouthKorea;Departmentof NewBiology,DGIST,Daegu,SouthKorea

HAN LI CellularPlasticityandDiseaseModeling,DepartmentofDevelopmentalandStem CellBiology,CNRSUMR3738,InstitutPasteur,Paris,France

NICOLAS MALAQUIN CentredeRechercheduCHUM(CRCHUM)andInstitutduCancer deMontre´al,Montreal,QC,Canada

AGGELOS MARGETIS MolecularCarcinogenesisGroup,DepartmentofHistologyand Embryology,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens, Greece

KENDRA I.MELOS DepartmentofMolecularMedicine,TheScrippsResearchInstitute, Jupiter,FL,USA

EMMANUEL MIKROS DivisionofPharmaceuticalChemistry,SchoolofPharmacy,National andKapodistrianUniversityofAthens,Athens,Greece;PharmaInformaticsUnit,Athena ResearchCenter,Athens,Greece

IORDANIS MOUROUZIS DepartmentofPharmacology,MedicalSchool,Nationaland KapodistrianUniversityofAthens,Athens,Greece

VASSILIOS MYRIANTHOPOULOS DivisionofPharmaceuticalChemistry,SchoolofPharmacy, NationalandKapodistrianUniversityofAthens,Athens,Greece;PharmaInformatics Unit,AthenaResearchCenter,Athens,Greece

LAURA J.NIEDERNHOFER InstituteontheBiologyofAgingandMetabolism,Universityof Minnesota,Minneapolis,MN,USA;DepartmentofMolecularMedicineandCenteron Aging,TheScrippsResearchInstitute,Jupiter,FL,USA

CONSTANTINOS PANTOS DepartmentofPharmacology,MedicalSchool,Nationaland KapodistrianUniversityofAthens,Athens,Greece

JOON TAE PARK DivisionofLifeSciences,CollegeofLifeSciencesandBioengineering, IncheonNationalUniversity,Incheon,SouthKorea

SANG CHUL PARK WellAgingResearchCenter,DGIST,Daegu,SouthKorea;TheFuture LifeandSocietyResearchCenter,ChonnamNationalUniversity,Gwangju,SouthKorea

JOAO F.PASSOS NewcastleUniversityInstituteforAgeing,NewcastleUniversity,Newcastle uponTyne,UK;InstituteforCellandMolecularBiosciences,NewcastleUniversity, NewcastleuponTyne,UK;AgeingResearchLaboratories,CampusforAgeingandVitality, NewcastleUniversity,NewcastleuponTyne,UK

ZIV PORAT FlowCytometryUnit,LifeSciencesCoreFacilities,WeizmannInstituteof Science,Rehovot,Israel

ARVIND RAMANATHAN BuckInstituteforResearchonAging,Novato,CA,USA

SOPHIA V.RIZOU MolecularCarcinogenesisGroup,DepartmentofHistologyand Embryology,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens, Greece

PAUL D.ROBBINS InstituteontheBiologyofAgingandMetabolism,Universityof Minnesota,Minneapolis,MN,USA;DepartmentofBiochemistry,MolecularBiologyand Biophysics,UniversityofMinnesota,Minneapolis,MN,USA;DepartmentofMolecular MedicineandCenteronAging,TheScrippsResearchInstitute,Jupiter,FL,USA

FRANCIS RODIER CentredeRechercheduCHUM(CRCHUM)andInstitutduCancerde Montre´al,Montreal,QC,Canada;De´partementdeRadiologie,Radio-OncologieetMe ´ decineNucle´aire,Universite´deMontre´al,Montreal,QC,Canada

CLEMENS A.SCHMITT DepartmentofHematology,OncologyandTumorImmunology, MolekularesKrebsforschungszentrum–MKFZ,Charite´–UniversityMedicalCenter,Berlin, Germany;DeutschesKonsortiumfu¨rTranslationaleKrebsforschung(GermanCancer Consortium),PartnerSiteBerlin,Berlin,Germany;Max-Delbru¨ck-CenterforMolecular Medicine,Berlin,Germany

PETROS SFIKAKIS FirstDepartmentofPropaedeuticInternalMedicineandRheumatology Unit,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens,Greece

RYAN K.SHULTZABERGER LeidosInc.,SanDiego,CA,USA

DIANA C.J.SPIERINGS EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA), UniversityofGroningen,UniversityMedicalCenterGroningen,Groningen,The Netherlands

I.SPIVAK InstituteofCytology,RussianAcademyofSciences,Saint-Petersburg,Russian Federation

NU ´ RIA TARRATS EdinburghCancerResearchUKCentre,InstituteofGeneticsand MolecularMedicine,UniversityofEdinburgh,Edinburgh,UK

AARON TAUDT EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA),University ofGroningen,UniversityMedicalCenterGroningen,Groningen,TheNetherlands; InstituteforComputationalBiology,HelmholtzZentrumMu¨nchen,Neuherberg,Germany

PAUL TOWNSEND FacultyInstituteforCancerSciences,ManchesterAcademicHealth SciencesCentre,UniversityofManchester,Manchester,UK

VE ´ RONIQUE TU CentredeRechercheduCHUM(CRCHUM)andInstitutduCancerde Montre´al,Montreal,QC,Canada

HILDAVANDEN BOS EuropeanResearchInstitutefortheBiologyofAgeing(ERIBA), UniversityofGroningen,UniversityMedicalCenterGroningen,Groningen,The Netherlands

PANAGIOTIS V.S.VASILEIOU MolecularCarcinogenesisGroup,DepartmentofHistologyand Embryology,MedicalSchool,NationalandKapodistrianUniversityofAthens,Athens, Greece

A.VASILISHINA InstituteofCytology,RussianAcademyofSciences,Saint-Petersburg, RussianFederation

STELLA VICTORELLI NewcastleUniversityInstituteforAgeing,NewcastleUniversity, NewcastleuponTyne,UK;InstituteforCellandMolecularBiosciences,Newcastle University,NewcastleuponTyne,UK;AgeingResearchLaboratories,CampusforAgeing andVitality,NewcastleUniversity,NewcastleuponTyne,UK

MATHIEUVON JOEST CellularPlasticityandDiseaseModeling,Departmentof DevelopmentalandStemCellBiology,InstitutPasteur,Paris,France

CHRISTOPHER D.WILEY BuckInstituteforResearchonAging,Novato,CA,USA

MATTHEW J.YOUSEFZADEH InstituteontheBiologyofAgingandMetabolism,Universityof Minnesota,Minneapolis,MN,USA;DepartmentofBiochemistry,MolecularBiologyand Biophysics,UniversityofMinnesota,Minneapolis,MN,USA;DepartmentofMolecular Medicine,TheScrippsResearchInstitute,Jupiter,FL,USA

LEI ZHANG DepartmentofMolecularMedicineandCenteronAging,TheScrippsResearch Institute,Jupiter,FL,USA

JING ZHAO DepartmentofMolecularMedicineandCenteronAging,TheScrippsResearch Institute,Jupiter,FL,USA;DiseaseBiologyandCellularPharmacology,Recursion Pharmaceuticals,SaltLake,UT,USA

DetectingCellularSenescenceinReprogramming

CoralieCazin,MathieuvonJoest,andHanLi

Abstract

Cellularsenescencehasbeensuggestedtofacilitatetissueregenerationviapromotingcellularplasticity. Here,wedescribemultiplesystems,bothinvitroandinvivo,todetectsenescenceinthecontextofcellular reprogramming.

Keywords Cellularsenescence,Reprogramming,Cellularplasticity,SA-β-Gal

1Introduction

Cellularsenescenceisastablecellcyclearrestcausedbystresses duringvariousbiologicalandpathologicalconditions[1–3].Interestingly,thesecellsremainmetabolicallyactiveandsecreteavast numberoffactorsincludingcytokines,chemokinesaswellas growthfactors,whichiscollectivelytermedasSASP(senescence associatedsecretoryphenotype).Senescentcellshavemultifaceted capabilitiesandareinvolvedinawiderangeofphysiologicaland pathologicalprocesses,suchasdevelopment,cancer,andaging [2, 4, 5].Morerecently,growingevidenceindicatesthatsenescent cellsmightfacilitatetissuerepairandregeneration[6, 7].

Cellularplasticityisthecapacityofacelltochangeitsidentity. Nuclearreprogrammingpresentsoneofthebestexamplesofcellularplasticity.Somaticcellscanbereprogrammedintoapluripotent stageviaforcedexpressionoftheYamanakafactors(Oct4,Sox2, Klf4,andc-Myc(OSKM)).Theinducedpluripotentstemcells (iPSCs)canbeobtainedbothinvitroandinvivo[8, 9].

Senescenceisimportantforcellularplasticity.Itisacellintrinsicbarrierforreprogramming[10].However,recentstudies suggestsenescentcellscouldpromotecellularplasticityextrinsically tofacilitatetissueregenerationviaSASPs[11–13]

MarcoDemaria(ed.), CellularSenescence:MethodsandProtocols,MethodsinMolecularBiology,vol.1896, https://doi.org/10.1007/978-1-4939-8931-7_1, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2019 CoralieCazinandMathieuvonJoestcontributedequallyandshouldbeconsideredco-firstauthors.

2Materials

2.1Generation ofSenescentCells

Takentogether,theemergingdatahighlightstheimportance ofinvestigatingcellularsenescence,particularlyinvivosenescence, inacontext-dependentmanner.Here,wepresentvariousprotocols toinvestigatetheimpactofcellularsenescenceoncellularplasticity, bothinvitroandinvivo.First,wewillintroducethesystemto studytheimpactofcellularsenescenceoninvitroreprogramming. Next,wewilldescribehowtodetectsenescentcellsintwotissues withdifferentsusceptibilitytoinvivoreprogramming:liver(permissive)andskeletalmuscle(nonpermissive)[11, 12].SA-β-Gal assayandantibodyimmunostainingareusedtogethertodetect senescentcells.Nanog,amarkerofpluripotency,isusedtoevaluate invivoreprogrammingintheliverandskeletalmuscle.

2.2InVitro Reprogramming

Prepareallthesolutionsusingsterilewater.Allthereagentsare preparedandstoredatroomtemperature(unlessindicated otherwise).

1.MouseEmbryonicFibroblasts(MEFs)[14].

2.Mouseembryofibroblast(MEF)Medium:Dulbecco’smodifiedEagleMedium(DMEM)withhighglucose(4.5g/L), 10%FBS,100U/mLpenicillin,and100 μg/mLstreptomycin.

3.Phosphate-bufferedsaline(PBS),autoclaved.

4.0.05%trypsin–EDTAsolution

5.1mg/mLmitomycinC(MMC)stocksolution,filteredand storedat 20 C.

6.X-rayirradiator(Optional).

7.0.2 μmfilters

8.Tissuecultureplates:100-mmand150-mm.

9.Conicalcentrifugetubes:15-mLand50-mL.

10.Centrifuge.

11.Phase-contrastinvertedlightmicroscope.

12.CO2 tissuecultureincubator.

13.Laminarflowhoodwithstandardtissueculturesetup.

1.Reprogrammingmedium:Dulbecco’smodifiedEagleMedium (DMEM)withhighglucose(4.5g/L),15%Knock-OutSerum Replacement(KSR),2mmGlutaMAX,0.1mmnonessential aminoacids,0.1mm2-mercaptoetanol,100U/mLpenicillin, and100 μg/mLstreptomycin,1000U/mLmouseleukemia inhibitoryfactor(LIF).

2.HEK293Tcells.

3.Wild-type(WT)MEFs.

2.4ARFandKi67 Staining

4.1mg/mLdoxycycline.

5.X-tremeGeneHPDNAtransfectionreagent(Roche).

6.Polybrenestocksolution(8mg/mL).

7.Retroviralvectors:pMXs-c-Myc,Addgene:13375;pMXsKlf4,Addgene:13370;pMXs-Sox2,Addgene:13367;pMXsOct3/4,Addgene:13366),pCL-Eco,Addgene:12371;controlretroviralvectorcontainingGFP.

8.Syringes.

9.0.45 μmfilters.

10.Aluminumfoil.

11.LabRocker.

12.4%paraformaldehyde(PFA)

13.Alkalinephosphatasedetectionkit.

1.SA-β-Galfixationsolution:2%formaldehydeand0.2%glutaraldehydeinPBS.

2.0.4mcitricacid/phosphatebuffer(pH ¼ 6.0):resuspend sodiumphosphatedibasic(Na2HPO4)andcitricacidmonohydrateinwater.Add36.85mLof0.1mcitricacidto 63.15mL0.2mdibasicsodiumphosphate.Mixandadjust pHto6withcitricacidifnecessary(see Note1).

3.X-Gal:DissolvetheX-Galpowderindimethylformamide (DMF)andstorein 20 C(see Note2).

4.X-Galsolution:40mmCitricacid/phosphatebuffer,150mm NaCl,2mmMgCl2 (StoreatRT.),4mmK3Fe(CN)6 (Storeat 4 C),4mmK4Fe(CN)6 ((Storeat4 C),1mg/mLX-Galin water,freshlymadeuponusageinatubewrappedwithaluminumfoil(see Note3).

5.X-Galsolution-musclespecific:4mmK3Fe(CN)6,4mmK4Fe (CN)6,2mmMgCl2,0.01%NP-40,and400 μg/mLX-Galin PBS,pH ¼ 5.5inatubewrappedwithaluminumfoil (see Note4).

6.0.2%(Eosin)(see Note5).

7.37 Cincubator.

1.PFAfixationsolution:PBScontaining4%paraformaldehyde.

2.Permeabilizationsolution:0.1%NaCitrate,0.5%TritonX-100 inwater(see Note4).

3.Blockingsolution:10%FBS,3%BSA,0.5%TritonX-100in PBS.Storedat4 C(see Note5).

4.PBS-0.5%Tween20:PBScontaining0.5%Tween20.

5.Antibodies:Ki67(Abcam,ab15580);p19Arf(SantaCruzBiotechnology,5-C3-1).

2.3SA-β-Gal Staining

2.5NANOGStaining

6.3,30 -diaminobenzidine(DAB)dilution:dilutethe3,30 -diaminobenzidine(DAB)inthebuffersolutionfromthekit (DAB+ +substratebuffer).20 μLofDABfor1mLofbuffer solution.

1.PFAfixationsolution:PBScontaining4%paraformaldehyde.

2.Permeabilizationsolution:0.1%NaCitrate,0.1%TritonX-100 inwater.StoreatRT.

3.Blockingsolution:5%FBSinPBS(see Note6).

4.Nanogantibody(CellSignaling,8822S).

5.EnVision+Kits(HRP.Rabbit.DAB+)DakoK4010.

2.6InVivo Reprogramming

1.Reprogrammablemousemodel[8].

2.Doxycycline(1mg/mL)(Sigma24390-14-5).

3.Cardiotoxin(LotaxanValence,France).Stocksolution (40 μm):1mgin3676 μLof0.9%NaCl,50 μL/aliquot, storeat 20 C.Workingsolution(10 μm):add150 μLof 0.9%NaClto50 μLstockaliquotoniceatthedayoftheinjury. Inject40 μL/TA.

4.0.3mLneedles:29G 1/200 –0.33 12mm.

3Methods

3.1Evaluation theImpactofCellular SenescenceonInVitro Reprogramming

3.1.1Generating SenescentMEFs

Caution:Allstepshavetobeperformedinasterileflowhood.

1.CultureandexpandMEFs:ThawonevialofMEFsinone 100-mmtissuecultureplate.Oncecellsareconfluent,pass themintoone150-mmtissuecultureplate.Passcellsagain intofive150-mmtissuecultureplates.Whencellsareconfluent,inducesenescenceeitherwithMMCtreatmentor irradiation.

2.MMCtreatmentinducedsenescence:Add1mg/mLMMC stocksolutiondirectlyintotheMEFmediumtoafinalconcentrationof10 μg/mL.TreatthecellswithMMCfor3hinthe incubator.

3.WashingthecellswithPBStwicetoremoveMMC.Trypsinize thecellsandresuspendtheminMEFmediumandcount.

4.Seedthecellsatthedensityof2.8 104 cells/cm2 (forexampleseed1.5 106 cellsin100-mmtissuecultureplate)and cultureinMEFmedium.Cellswillbecomesenescenceafter 48handcanbeconfirmedbySA-β-Galstaining.

3.1.2SA-β-GalStaining

5. γ-Irradiationinducedsenescence(Optional):After step1,cells canalsobetrypsinizedandresuspendedinMEFmedium, adjustingtheconcentrationto2–6 107 cells/mL.Gently mixthecellsandirradiatethemfortotal3000rad.Afterthe irradiation,cellscanbeuseddirectlyas step4,orkeptfrozen forfutureuse.

1.RemovemediumandwashMEFstwicewithPBS.Add SA-β-Galfixationsolutiontotheplateandmakesurethe solutioncoversthesurfacecompletely.Incubateatroomtemperaturefor15min.

2.AspiratethefixationsolutionandwashthreetimeswithPBS. IncubatecellswithfreshlymadeSA-β-Galsolutionovernightat 37 C,protectedfromlight.

3.Removethesolutioncompletelyandwashtheplateswith runningwater.PlatescanbestoredinPBSat4 Cupto 1week,protectedfromlight.

3.1.3InVitro

Reprogramming withSenescenceConditionalMedium

1.GeneratingsenescenceconditionalMedium(CM):Incubate senescentcellswithreprogrammablemediumw/oLIF (10mLmediumfor100-mmplate).CollecttheCMevery 24handreplacewith10mLfreshreprogrammablemedium w/oLIF.CMcanbecollectedfor5days.Filterthecollected CMusing0.2 μmfilter.CMcanbeuseddirectlyorkeptat 20 C.

2.ReprogrammingMEFswithretroviralinfection:invitroreprogrammingisperformedasdescribedpreviously[15].Day1: Seed5 106 293Tcellsinone100-mmplate.

3.Day2:Transfect293TcellsusingX-tremeGeneHPtransfectionreagentandpMXs-vectors.Mix4 μgofindividualpMXs plasmidorcontrolvector(e.g.,pMSCVPuroIRESGFP)with 4 μgofpCLEco.Incubatetheplasmidsmixwith8 μLof X-tremeGeneHPtransfectionreagentand594 μLof DMEM(DNA:transfectionreagent ¼ 1:1)atRTfor30min. Addoneplasmidsmixontooneplateof293Tcells.

4.Day3:ChangethemediumofHEK293TcellsusingMEF medium.Onthesameday,seed5 105 WTMEFs/100-mm plateinMEFmedium.

5.Day4–5:retrovirusinfectionofMEFs.Collectmediumfrom every293Tplateinseparatefalcontubesandreplacewith 10mLoffreshMEFmedium.Centrifugethecollected mediumat250 g for5minatRT.Passthemediumthrough 0.45 μmfiltersandaddPolybrenetothefinalconcentrationof 8 μg/mL.Mixthefactors(2mLofeveryfactor/plate)firstina falcontubethenaddthemixontoWTMEFs.Performfour roundsofinfectionintotal,12hinterval.

3.2Evaluation theImpactofCellular SenescenceonInVivo Reprogramming

3.2.1SA-β-GalStaining onFrozenLiverSection

6.Day6:SeedtheinfectedMEFsonto35-mmplatesinMEF medium.TheamountofMEFsseededshouldyield20–40 clonesperplate,whichdependentontheinfectionandreprogrammingefficiency.Itisadvisedtodeterminetheseparameterspriortotheexperiment.

7.Day7:ReplacethemediumtoCMmediumsupplemented withLIF(1000U/mL)tostartreprogramming.

8.Changethemediumevery2days.iPSCscoloniesshouldbe clearlyvisibleunderthemicroscopeafter2weeks.

9.QuantificationofiPSCs:Oncethecoloniesareclearlyvisible, theplatesareprocessedforalkalinephosphatase(AP)staining accordingtothemanufacturer’sprotocol.Quantificationcan bedoneeithermanuallyorwithimageJsoftware.

1.Fixthesectionsfor4mininfixationsolution,atRT(see Note 7).WashthesectionswithPBSthreetimes,5mineachtime.

2.IncubatethesectionsintheX-galsolutionat37 Covernight (see Note8).WashthesectionswithPBSthreetimes,10min eachtime.

3.Post-fixedin1%paraformaldehydeinPBSfor30min,atRT (see Note7).WashthesectionswithPBSthreetimes,10min eachtime.

4.MounttheslideswithPBScontaining20%glycerol.

3.2.2SA-β-GalStaining onFrozenMuscle Section[16]

Thetibialisanterior(TA)musclesofreprogrammablemiceare injuredwithcardiotoxinandtreatedwithdoxycycline(1mg/mL) inthedrinkingwaterfor7daystoinducebothsenescenceand reprogramminginvivo.TAmusclesareharvestedandpreparedas describedelsewhere[16].

1.Fixthesectionsfor4mininfixationsolution,atRT(see Note 7).WashthesectionswithPBSthreetimes,5mineverytime.

2.Incubatesectionsfor30mininPBSpH ¼ 5.5(see Note9).

3.IncubatesectionsintheX-galsolutionmusclespecificat37 C foratleast24hprotectedfromlight(see Note10).Washthe sectionswithPBSthreetimes,10mineverytime.

IfonlySA-β -Galstainingisdesired,continuewiththenextsteps. IfcostainingwithKi67isdesired,pleaseforwardtoSubheading 3.2.4.IfcostainingwithNanogisdesired,pleaseforwardtoSubheading 3.2.5

4.Post-fixin1%paraformaldehydeinPBSfor30min,atRT(see Note7).WashthesectionswithPBSthreetimes,10min eachtime.

3.2.3Immunostaining UsingAnti-p19ARF

5.Counterstainwith0.2%eosinatRT.Immersetheslidesinthe eosinsolutionfor1minandrinsethemwithwaterbriefly(see Note11).

6.MounttheslideswithPBScontaining20%glycerol(see Note12).

7.Post-fixin1%paraformaldehydeinPBSfor30min,atRT(see Note7).WashthesectionswithPBSthreetimes,10min everytime.

8.Counterstainwith0.2%eosinatRT.Immersetheslidesinthe eosinsolutionfor1minandrinsethemwithwaterbriefly(see Note11).

9.MounttheslideswithPBScontaining20%glycerol(see Note12).

1.FixtheslideswithPFAfixationsolutionfor10minatRT(see Note7).WashthesectionswithPBSthreetimes,10min eachtime.

2.Add200 μLofthepermeabilizationsolutiondirectlyontothe slidesandincubateatRTfor5min.Washthesectionswith PBS-Tween20twice,5mineachtime.

3.Add200 μLofblockingsolutiondirectlyontheslidesfor 30minatRT.

4.Incubatewiththeprimaryantibodies:2 μg/mLofKi-67or 0.8 μg/mLofp19Arfovernightat4 Cintheblockingsolution(see Note13).WashthesectionswithPBS,10min eachtime.

5.Washwith200 μLPBScontaining0.25%BSAonslidesatRT for5min(see Note14).

6.Incubatewiththesecondaryantibodyinblockingsolutionfor 1hatRT(see Note15).WashthesectionswithPBSforthree times,5mineachtime.

7.Mounttheslideswithaqueousnonfluorescingmounting medium.

3.2.4ImmunohistochemistryUsing Anti-Ki67

1.FixtheslideswithPFAfixationsolutionfor10minatRT(see Note7).WashthesectionswithPBSthreetimes,10min eachtime.

2.Add200 μLofthepermeabilizationsolutiondirectlyontothe slidesandincubateatRTfor5min.Washthesectionswith PBS-0.5%Tween20twice,5mineachtime.

3.Add200 μLofblockingsolutiondirectlyontheslidesfor 30minatRT.

4.Adding100 μLofrAb-HRPfromDakokit(readytouse)for 45minatRT(see Note15).WashthesectionswithPBSthree times,5mineachtime.

5.DiluteDABinthebuffersolution(see Note16).

3.2.5ImmunohistochemistryUsing Anti-NANOGAntibody onFrozenTissueSections

6.Visualization:add100 μLofDABpreviouslydiluted(see Note 16)oneveryslideupto10minatRT.Observetheslidesunder themicroscope(see Note17).Stopthereactionbywashing withwater.

1.FixtheslideswithPBScontaining4%paraformaldehydefor 10min,atRT(see Note7).WashthesectionswithPBStwice, 10mineachtime.

2.Add200 μLofthepermeabilizationsolutiondirectlyontothe slidesandincubateatRTfor5min.WashthesectionswithPBS twice,5mineachtime.

3.Washthesectionswith200 μLPBScontaining0.25%BSA directlyonslidesatRTfor5min(see Note14).

4.Incubatetheslideswith1.25 μg/mLofNanogantibodyovernightat4 CinPBScontaining5%FBS(see Note15).Wash thesectionswithPBStwice,10mineachtime.

5.Washwith200 μLPBScontaining0.25%BSAontheslidesat RTfor5min(see Note14).

6.Incubatewiththesecondaryantibodybyadding100 μLof rAb-HRPfromDakokitfor45minatRT(see Note15).Wash thesectionswithPBSthreetimes,5mineachtime.

7.DiluteDABinthebuffersolution(see Note16).

8.Visualization:add100 μLofDABsolutiononeveryslideup to10minatRT.Observetheslidesunderthemicroscope(see Note17).Stopthereactionbywashingwithwater.

9.CounterstainwithFastredsolutionfor20min,atRT(see Note11).Washwithwaterbriefly.

10.Dehydratewith95%ethanolfor5minfollowedwith100% ethanol,2 5min.

11.Mounttheslideswithquick-hardeningmountingmedium.

4Notes

1.Thecitricacid–phosphatebuffercanbestoredat4 C.AdjustingthepHisacrucialstepforstaining.

2.TheXgalcanbestoredinaliquot,protectedfromlight,at 20 Cupto6months.

3.TheK3Fe(CN)6 solutionandK4Fe(CN)6 solutioncanbe storedat4 Cbuttheyneedtobeprotectedfromlight.

4.Wefindthatthissolutionworksbetterforthemuscle cryosections.

References

5.EosinsolutioncanbekeptatRTandreusedafterfilteringif necessary.

6.Blockingsolutionscanbefilteredthrougha0.45 μmfilter, aliquotedandstoredat 20 C.Itcanbestoredat4 Cfor 6months.

7.Performthefixationunderthehood.Donotfixlongerto maintainaproperstainingandlettheenzymaticreactions occurforaproperSA-β-Galstaining.Itisessentialtoperform thepost-fixationforSA-β-Galstainingaloneforagoodconservationofthestaining.

8.Makesurethatthetemperatureisat37 Candthatslidesare protectedfromlightovernight.

9.AdjustingthepHisacrucialstepforstaining.Useamagnetic stirbartoobtainthecorrectpHofthefinalsolution.

10.Theincubationrequiresminimal24handcanlastfor48hto maximizetheSA-β-Galsignal.Thesolutionneedstobe changedafter24hincubation.

11.EosinsolutionandfastredsolutioncanbekeptatRTand reusedafterfilteringifnecessary.Incubationtimecanbe adjusteddependingontheintensitywanted.Slidesshouldbe analyzedquicklyaftermountingforeosincounterstaining becausetheeosinissolubleinwaterandthecounterstaining willbeweakerwithtime.WechooseeosinbecauseSA-β-Gal stainingisnotstableinwater.

12.Foralongerconservation,youcanmounttheslideswith aqueousnonfluorescingmountingmedium.

13.Incubateslidesinaboxwithwetpapertoweltoprevent evaporation.

14.Wefindthatitisbesttopreparethisfresheachtime.

15.Incubateslidesinaboxwithwetpapertoweltoprevent evaporationandprotectfromlight.

16.FreshlypreparedandthedilutedDABsolutionisstableupto 1weekat4 C.

17.Theincubationtimecanbeadjustedtominimizethebackgroundbuthavetobethesameforalltheslides.

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DNADamageInSituLigationFollowedbyProximity LigationAssay(DI-PLA)

AlessandroGalbiatiandFabriziod’AddadiFagagna

Abstract

CellshaveevolvedDNArepairmechanismstomaintaintheirgeneticinformationunalteredandaDNA damageresponsepathwaythatcoordinatesDNArepairwithseveralcellularevents.Despiteaclearrolefor DNAdamageintheformofDNAdouble-strandbreaks(DSBs)inseveralcellularprocesses,themost commonlyusedmethodstodetectDNAlesionsareindirect,andrelyonantibody-basedrecognitionof DNAdamage-associatedfactors,leavingseveralimportantquestionsunanswered.Differently,herewe describeDNAdamageInsituligationfollowedbyProximityLigationAssay(DI-PLA),thatallowssensitive detectionofphysicalDSBsinfixedcells,throughdirectlabelingoftheDSBswithbiotinylatedoligonucleotides,andsubsequentsignalamplificationbyPLAbetweenbiotinandapartnerproteinintheproximity oftheDNAbreak.

Keywords DI-PLA,PLA,Single-cell,Imaging,DNAdamageresponse(DDR),DNAdamage,DNA double-strandbreak(DSB)

1Introduction

Genomesareconstantlysubjectedtoaplethoraofstimulithatcan leadtoDNAdamage,which,inturn,altersthegenomicstructure andthegeneticinformationencodedwithin.Amongthedifferent typesofDNAlesions,DNAdouble-strandbreaks(DSBs)areparticularlycytotoxic,because,ifunrepairedbeforecelldivision,can leadtothelossofcriticalgeneticinformationandtochromosomal rearrangements.Inordertorepairdifferentkindoflesions,cells havedevelopedfinelytunedDNArepairmechanismsandasignalingcascadeknownasDNAdamageresponse(DDR)thatcoordinatesseveralcellularevents,includingatransientarrestofcell proliferationtopreventthepropagationofalteredgenomicinformationtothedaughtercells[1].DNAdamageaccumulationhasa keyroleinpathologicaleventssuchascanceronsetandorganismal aging[2],cellulardifferentiation[3],cellularreprogramming[4], andtranscriptionmodulation[5].

MarcoDemaria(ed.), CellularSenescence:MethodsandProtocols,MethodsinMolecularBiology,vol.1896, https://doi.org/10.1007/978-1-4939-8931-7_2, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2019

DespitetheimportanceofDNAdamage,thewell-established methodstodetectDNAdamageaccumulationincellsandtissues (immunostainingandchromatinimmunoprecipitation—ChIP)are basedonantibodyrecognitionofchromatinmodificationsassociatedwithDNAdamageorofproteinsinvolvedinDDRsignaling orrepair.ThisapproachmightresultinartifactualDNAdamage detection,sincesomereportssuggestthatuncouplingbetween DDRmarkersaccumulationandDNAdamagepresenceispossible underparticularcircumstances.Forexample,ithasbeenobserved thatheterochromaticregionsarepartiallyresistanttoDDRmarkers accumulation[6],andthatsomecelltypes,suchasastrocytes,lacka fullDDRactivationinthepresenceofDNAdamage[7].Viceversa, activationofDDRintheabsenceofphysicalDNAdamageisalso possible[8].

AfewalternativestoimmunostainingandChIPforDDR markersareavailable.Recently,severalmethodshavebeendevelopedtodirectlymapDSBaccumulationatgenome-widelevelwith single-nucleotideresolution[5].However,thesemethodsarelimitedbytheirlowsensitivityandcanonlybeappliedtodetect recurringDSBsinapopulationofcells.Instead,tostudyDNA damagepresenceinsingle-cells,theonlyalternativestoimmunofluorescenceareterminaldeoxynucleotidetransferasedUTPnick endlabeling(TUNEL)[9]andCOMETassay[10].TUNELrelies ontheenzymaticmodificationofexposedDNAends,withthe additionofbiotinylateddNTPs:thisallowsphysicaldetectionof DSBsusingwithfluorophore-conjugatedanti-biotinantibodies. Differently,intheCOMETassay,asuspensionofcellsismixed withagaroseandspreadontoamicroscopeglassslide.Cellsare thenlysedandDNAunwindingbyelectrophoresisiscarriedoutat neutraloralkalinepH.Whensubjectedtoanelectricfield,the DNAfragmentsmigrateoutofthenucleoid,towardtheanode, appearinglikea“comet”:thesizeandshapeofthecometandthe distributionofDNAwithinthecometcorrelatewiththeextentof DNAdamage[11].However,bothmethodshavelowsensitivity, thustheirapplicationisusuallylimitedtostudymassiveDNA damage,asinthecaseofcellscommittingtoapoptosis;moreover, TUNELcannotdistinguishbetweensinglestrandbreaks(SSBs) andDSBs,whileCOMETcannotbeeasilyappliedincombination withotherimmunostainings.

Herewedescribearecentlypublishedmethod,DNAdamage Insituligationfollowedbyproximityligationassay(DI-PLA),to detectphysicalDSBsinproximityofatargetproteininindividual cells(Fig. 1).Briefly,adherentcellsaregrownonglasscoverslips, fixed,andpermeabilizedtoallowenzymaticmodificationofgenomicDNA.Specifically,exposedgenomicDNAendsofDSBsare bluntedinsituandligatedtoadouble-strandhairpin-shapedbiotinylatedDNAoligonucleotide,whichpermanentlytagsallDSBs inthecell.Then,proximityligationassay(PLA)[12],isperformed

Cell fixationand permeabilization

DNA endsblunting and ligationto the biotinylatedlinker

Incubationwith primaryantibodies againstbiotinand a proteinof interest

Fig.1 DI-PLAworkflow.Cellsarefirstfixedinparaformaldehydeandpermeabilized.Then,exposedDNAends arebluntedinsituandligatedtohairpin-shapedbiotinylatedDNAoligonucleotides(green).Next,cellsare incubatedwithantibodiesraisedagainstbiotin(red)andapartnerproteinintheproximity(lightblue)ofthe break.Finally,PLAproducesfluorescentsignals(yellowdots)atthesiteofbreak,whichcanbedetectedby microscopy

usinganantibodyrecognizingthebiotinandapartnerantibody raisedagainstaproteinintheproximityofthebreak,suchasaDDR marker.Withthisstrategy,itispossibletoobtainsingle-molecule sensitivity,requiredtodetectthebiotinontheDNAoligonucleotidetaggingtheDSB,whichwouldbeotherwiseundetectableby standardimmunostainingwithananti-biotinantibody.

Byexploitingthistechniqueweprovedthepresenceofphysical DSBsintheproximityofactivatedDDRmarkersinsenescentcells

[13].Interestingly,besidesprobingforthepresenceofphysical DSBsintheproximityofDDRfactors,DI-PLAcouldalsobe adaptedtodetectDSBatspecificlocithatcanbeidentifiedbya specificsetofproteins,suchascentromeresortelomeres.Furthermore,itispossibletouse,asbiotinpartner,awidespreadchromatin marker,suchasacorehistone,toachieveDNAdamagedetectionin singlecellswithoutrelyingonanyDDRmarker.

2Materials

2.1Solutions

2.2Dedicated Reagents

Prepareallsolutionsusingpure,deionizedwaterandmolecular biologygradereagents.Dithiothreitol(DTT)-containingsolutions (NEB2buffer,bluntingbuffer,andligationbuffer)shouldbe preparedfreshdaily.

1.Fixingsolution:paraformaldehyde(PFA),4%inDulbecco’s phosphatebufferedsaline(DPBS)1

2.Permeabilizationbuffer:DPBS1 ,0.2%TritonX-100(see Note1).

3.NEB2Buffer:50mMNaCl,10mMTris–HCl,10mM MgCl2,1mMDTT,0.1%TritonX-100,pH8at25 C.

4.Bluntingbuffer:100mMTris–HCl,50mMNaCl,10mM MgCl2,5mMDTT,0.025%TritonX-100pH7.5at25 C.

5.Ligationbuffer:50mMTris–HCl,10mMMgCl2,1mMATP, 10mMDTT,pH7.5at25 C.

6.Blockingbuffer(PBG):0.5%BSA,0.2%gelatinfromcoldfish inDPBS;(see Note2).

7.PLAhybridizationmix:diluteDuolinkInsituPLAProbes PLUSandMINUS(Sigma)totheworkingconcentration1 inDuolinkAntibodyDiluent(Sigma);(see Note3).

8.PLAligationmix:diluteDuolinkLigationbufferandDuolink Ligase(Sigma)totheworkingconcentration1 inpurewater; (see Note4).

9.PLAamplificationmix:diluteDuolinkAmplificationbuffer andDuolinkPolymerase(Sigma)totheworkingconcentration 1 inpurewater;(see Note5).

10.DAPI:40 -6-Diamidino-2-phenylindole0.2 μg/mLinDPBS; (see Note6).

1.Bluntingenzyme:providedintheQuickBluntingkit(NEB, Cat.No.E1201L).

2.T4Ligase,400,000U/mL(NEB,Cat.No.M0202L).

3.Mowiolorsimilarmountingmedia.

4.DuolinkWashbuffer(Sigma,catNo.DUO82049)(see Note7).

2.3Antibodies

2.4Dedicated Equipment

3Method

3.1Fixationand Permeabilization

5.DI-PLALinker:DNAoligonucleotidedilutedinwaterwith thefollowingsequence(see Note8):

IDSequence DI-PLA Linker

1.Anti-biotin,rabbit(Abcam,Cat.No.AB53494),workingdilution1:2000.

2.Anti-biotin,mouse(Sigma,Cat.No.B7653),workingdilution 1:2000.

Thepartnerantibodyusedincombinationwiththeanti-biotin antibodyisdependentonDI-PLAapplication.Here,wesuggest twovalidatedDDRantibodiesthatcanbeusedincombination withbiotinasbenchmarks:

3.Anti-γH2AX,mouse(Millipore,Cat.No.05-636),working dilution1:2000.

4.Anti-53BP1,rabbit(Bethyl,Cat.No.A300-272A),working dilution1:2000.

1.24-wellplasticmultiwells.

2.Glasscoverslips.

3.Glassslides.

4.Tweezers(see Note9).

5.Parafilm.

Carryoutallproceduresatroomtemperature(RT),unlessotherwisespecified.For13mMcoverslips(see Note10),putthecoverslipsina24-wellplateandperformallwasheswithatleast300 μL ofbuffer.

Allenzymaticreactionsandantibodiesincubationsarecarried outinahumidifiedsealedchamber(see Note11).Growadherent cellsontheglasscoverslips.

1.Afterthetreatmentofinterest,cellsarefixedwiththefixing solutionfor10min.

2.CoverslipsarewashedtwiceinDPBS,thenareincubatedfor 10mininthepermeabilizationbuffer(see Note12).

3.CoverslipsarewashedtwiceinDPBSfor5min.

3.2DNAEnds BluntingandLinker Ligation

3.3Blockingand Incubationwith PrimaryAntibodies

3.4Proximity LigationAssay

1.CoverslipsarewashedtwiceinNEB2bufferfor5min.

2.Coverslipsarewashedtwiceinbluntingbufferfor5min.

3.Prepare35 μLofBluntingreactionmixforeachcoverslip: 1mMdNTPs,3.5 μLBluntingBuffer10 ,0.2mg/mL BSA,0.7 μLBluntingEnzyme,addH2Otoreachthefinal volume.Spot35 μLofbluntingreactiononParafilmand incubateeachcoverslip,withcellsfacingthereactionmix(see Note13),for1hinahumidifiedsealedchamber.

4.CoverslipsarewashedtwiceinNEB2bufferfor5min.

5.CoverslipsarewashedtwiceinLigationBufferfor5min.

6.Prepare50 μLofLigationreactionforeachcoverslip:0.5 μM DI-PLALinker,5 μLLigationbuffer10 ,1mMATP, 0.2mg/mLBSA,1.5 μLT4Ligase,addH2Otoreachthe finalvolume.Spot50 μLofligationreactiononParafilmand incubateeachcoverslip,withcellsfacingthereactionmix(see Note13),inahumidifiedsealedchamber,overnightat16 C.

1.CoverslipsarewashedtwiceinDPBSfor10min.

2.Coverslipsareblockedfor1hin500 μLPBGatRT.

3.Prepareprimaryantibodiesmix(see Note14).Spot50 μLof antibodymixonParafilmandincubateeachcoverslip,with cellsfacingthemixinahumidifiedsealedchamberfor1hat RTorovernightat+4 C.

1.CoverslipsarewashedtwiceinPBGfor5min.

2.Prepare35 μLofPLAhybridizationmixforeachcoverslip. SpotthemixonParafilmandincubateeachcoverslip,withcells facingthemix,inadarkhumidifiedchamberfor1hminat 37 C.

3.CoverslipsarewashedtwiceinDuolinkWashbufferA1 for 5min.

4.SpotthemixonParafilmandincubateeachcoverslip,withcells facingthemix(see Note13),inadarkhumidifiedchamberfor 30minat37 C.

5.CoverslipsarewashedtwiceinDuolinkWashbufferA1 for 2min.

6.Prepare35 μLofPLAAmplificationmixforeachcoverslip. SpotthemixonParafilmandincubateeachcoverslip,withcells facingthemix(see Note13),inadarkhumidifiedchamberfor 90minat37 C.

7.CoverslipsarewashedtwiceinDuolinkWashbufferBfor 10min.

8.CoverslipsareincubatedinDAPIfor3min.

Fig.2 TypicalDI-PLAoutput.(a)U2OScellseitheruntreatedorexposedto2Gyionizingradiationandfixed1h afterirradiation.DI-PLAwithantibodiesagainstbiotinand γH2AX.DNAstainedbyDAPI.Quantificationsare showninpanel(b)

9.CoverslipsarewashedinDPBS.

10.Coverslipsarewashedinpurewatertoremoveanyremaining salts.

11.Coverslipsaredriedandmountedonmicroscopeglassslides withMowiolorsimilarmountingmedia.

12.LetcoverslipsdryinthedarkforafewhoursatRTbefore acquiringimagesatthemicroscope(see Note15).

3.5ImagingAnalysis

1.Performimageacquisitionwithafluorescentmicroscope,ata suggestedmagnificationof20–60 .Foreachfieldacquirethe 408channel(tovisualizethenucleistainedbyDAPI)andthe appropriatechannelcorrespondingtotheDuolinkdetection reagentused(forexampleacquireCy3iftheOrangedetection reagenthasbeenused);(see Note16).Tocomparedifferent experimentalconditions,acquireallimageswithidenticalparameters.AtypicalDI-PLAexperimentresultisshowinFig. 2

2.QuantifythenumberofDI-PLAdotsineachnucleus,usingan imagingsoftwaresuchasCellProfiler[14];(see Note17).

4Notes

1.Prepareastocksolutionof10%TritonX-100dilutedinpure water.ThestocksolutioncanbestoredatRT.

2.Prepareastocksolutionof10 PBGandstoreat 20 C. Avoidfreeze—thawcycles.Theworkingsolution1 PBGcan bestoredat+4 Cforafewdays.

3.DuolinkprobesaresecondaryantibodiesconjugatedwitholigonucleotidesthatareusedtoprimethePLAreaction.Usea

combinationofDuolinkInsituPLAprobesPLUSandMINUS asneeded.Theanti-biotinantibodyandthepartnerantibody againstaproteinofinterestmustberaisedindifferentspecies. Forexample,ifananti-biotinantibodyraisedinrabbitisusedin combinationwithananti-γH2AXantibodyraisedinmouse,a combinationofMousePLUS(Sigma,catNo.DUO92001) andRabbitMINUS(Sigma,catNo.DUO92005)probes mustbeused.Probesareavailablealsoforantibodiesraisedin goat.Preparethemixinice,justbeforeuse.

4.ThePLAligationreactionaddsaconnectoroligonucleotideto oneoftheDuolinkprobestogenerateasinglestrandDNA circlethatservesastemplateforthesubsequentPLAreaction. ThePLAligationreagentsarepartoftheDuolinkdetection reagents.Preparethemixinice,justbeforeuse.

5.TheDuolinkamplificationreactionsisarollingcircleamplification(RCA)reactionthatisprimedbyoneofDuolinkprobes anduses,astemplate,thecirclestrandDNAcircleligatedto theotherDuolinkprobe.Duetospatialconstraints,thereactioncanonlytakeplacewherethetwotargetproteinsarein closeproximity(<40nm).Togenerateadetectablesignal,the Duolinkamplificationbuffercontainsfluorescentconjugated probesthathybridizetotheRCAproduct.Accordingtothe Duolinkdetectionreagentchosen,itispossibletogenerate fluorescentDI-PLAsignalsinthecolorofinterest.Thisisa featurethatmayproveusefulwhendoingacostainingwith immunofluorescenceforaparticularprotein,orwhenusing cellsexpressingfluorescence-taggedproteins.However,we observedthattheOrangedetectionreagent(Sigma,Cat. No.DUO92007)providesthebestsignaltonoiseratioand ismoreresistanttophotobleachingcomparedtotheother availabledetectionreagents.Preparethemixinicejust beforeuse.

6.Storeinthedarktoavoidfading.

7.StoretheWashbuffersat+4 C,upto6months.Itiscriticalto equilibratethewashbufferatRTbeforeuse.

8.DI-PLAlinkercanbeorderedwithastandarddesaltpurificationmethod.HPLCorSDS-PAGEpurificationmethodsyield apurerlinkerpopulation,thustheamountofoligousedfor eachligationreactioncanbereducedupto10times.Dilutein purewatertoobtaina100 μMstocksolution.Avoidrepeated freeze–thawcycles.

9.Tweezersareusedtohandlethecoverslips.

10.Iflargerglasscoverslipsareused,washingandreaction volumesmustbescaledupproportionally.

11.Itiscriticalthatthehumidifiedchamberiswellsealedandwell humidifiedtoavoidevaporationofthereactionmixes.

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Tresilian, Robert, C. T. i. 162.

Tribulus (Nicholas Brembre), C. T. i. 154.

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Walsingham’s Chronicle referred to, i. 855, 941, 1173, iv. 723, 959, C. T. i. 65, 80, 142, 150, 154, iii. 256, 394, 432.

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THE END

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