EMBO reports - Peer Review Process File - EMBO-2016-42360
Manuscript EMBO-2016-42360
Proteasomal Inhibition Selectively Kills Stem Cells in
Glioma-Derived Cancer Cells Through Endoplasmic
Reticulum-Associated Apoptosis
Young Dong Yoo, Hyunjoo Cha-Molstad, Ki Sa Sung, Dae-Hee Lee, Seung A Choi, Joonsung
Hwang Hwang, Ms. Su Ran Mun, Mr. Changhoon Ji, Deric Park, Seung Ki Kim, Yong Lee, Aaron
Ciechanover, Bo-Yeon Kim
Corresponding author: Yong Tae Kwon, Seoul National University College of Medicine
Review timeline:
Submission date:
Accepted:
11 March 2016
09 November 2016
Editor: Achim Breiling
No Peer Review Process File is available with this article, as the authors have chosen not to make the review
process public in this case.
© European Molecular Biology Organization
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CorrespondingAuthorName:YongTaeKwon
JournalSubmittedto:EMBOReports
ManuscriptNumber:EMBOR-2016-42360V1
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ReportingChecklistForLifeSciencesArticles(Rev.July2015)
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A-Figures
1.Data
Thedatashowninfiguresshouldsatisfythefollowingconditions:
è thedatawereobtainedandprocessedaccordingtothefield’sbestpracticeandarepresentedtoreflecttheresultsofthe
experimentsinanaccurateandunbiasedmanner.
è figurepanelsincludeonlydatapoints,measurementsorobservationsthatcanbecomparedtoeachotherinascientifically
meaningfulway.
è graphsincludeclearlylabelederrorbarsforindependentexperimentsandsamplesizes.Unlessjustified,errorbarsshould
notbeshownfortechnicalreplicates.
è ifn<5,theindividualdatapointsfromeachexperimentshouldbeplottedandanystatisticaltestemployedshouldbe
justified
è SourceDatashouldbeincludedtoreportthedataunderlyinggraphs.Pleasefollowtheguidelinessetoutintheauthorship
guidelinesonDataPresentation.
http://grants.nih.gov/grants/olaw/olaw.htm
http://www.mrc.ac.uk/Ourresearch/Ethicsresearchguidance/Useofanimals/index.htm
NIHGuidelinesinanimaluse
MRCGuidelinesonanimaluse
http://ClinicalTrials.gov
ClinicalTrialregistration
http://www.consort-statement.org
CONSORTFlowDiagram
http://www.consort-statement.org/checklists/view/32-consort/66-title
CONSORTCheckList
http://www.equator-network.org/reporting-guidelines/reporting-recommendations-for-tumour-marker-prognostic-studies-remark/
REMARKReportingGuidelines(markerprognosticstudies)
2.Captions
Eachfigurecaptionshouldcontainthefollowinginformation,foreachpanelwheretheyarerelevant:
è
è
è
è
aspecificationoftheexperimentalsysteminvestigated(egcellline,speciesname).
theassay(s)andmethod(s)usedtocarryoutthereportedobservationsandmeasurements
anexplicitmentionofthebiologicalandchemicalentity(ies)thatarebeingmeasured.
anexplicitmentionofthebiologicalandchemicalentity(ies)thatarealtered/varied/perturbedinacontrolledmanner.
http://datadryad.org
Dryad
http://figshare.com
Figshare
http://www.ncbi.nlm.nih.gov/gap
dbGAP
http://www.ebi.ac.uk/ega
EGA
http://biomodels.net/
BiomodelsDatabase
http://biomodels.net/miriam/
http://jjj.biochem.sun.ac.za
http://oba.od.nih.gov/biosecurity/biosecurity_documents.html
http://www.selectagents.gov/
MIRIAMGuidelines
JWSOnline
BiosecurityDocumentsfromNIH
ListofSelectAgents
è theexactsamplesize(n)foreachexperimentalgroup/condition,givenasanumber,notarange;
è adescriptionofthesamplecollectionallowingthereadertounderstandwhetherthesamplesrepresenttechnicalor
biologicalreplicates(includinghowmanyanimals,litters,cultures,etc.).
è astatementofhowmanytimestheexperimentshownwasindependentlyreplicatedinthelaboratory.
è definitionsofstatisticalmethodsandmeasures:
Ÿ commontests,suchast-test(pleasespecifywhetherpairedvs.unpaired),simpleχ2tests,WilcoxonandMann-Whitney
tests,canbeunambiguouslyidentifiedbynameonly,butmorecomplextechniquesshouldbedescribedinthemethods
section;
Ÿ aretestsone-sidedortwo-sided?
Ÿ arethereadjustmentsformultiplecomparisons?
Ÿ exactstatisticaltestresults,e.g.,Pvalues=xbutnotPvalues<x;
Ÿ definitionof‘centervalues’asmedianoraverage;
Ÿ definitionoferrorbarsass.d.ors.e.m.
Anydescriptionstoolongforthefigurelegendshouldbeincludedinthemethodssectionand/orwiththesourcedata.
Pleaseensurethattheanswerstothefollowingquestionsarereportedinthemanuscriptitself.Weencourageyoutoincludea
specificsubsectioninthemethodssectionforstatistics,reagents,animalmodelsandhumansubjects.
Inthepinkboxesbelow,providethepagenumber(s)ofthemanuscriptdraftorfigurelegend(s)wherethe
informationcanbelocated.Everyquestionshouldbeanswered.Ifthequestionisnotrelevanttoyourresearch,
pleasewriteNA(nonapplicable).
B-Statisticsandgeneralmethods
Pleasefillouttheseboxesê(Donotworryifyoucannotseeallyourtextonceyoupressreturn)
1.a.Howwasthesamplesizechosentoensureadequatepowertodetectapre-specifiedeffectsize?
Forallstatistics,atleastthreeindependentexperimentswereperformedtoearnpointvalue
representingmeanstandarddeviation.Statisticalanalysiswereperformedusingtwo-sided,one
typestudentT-TEST.p-valuebiggerthan0.05wasconsideredassignificantvalue.Forlivecell
countingexperiments,atleast50cellsfromrandomlychosenfieldswereblindlycounted.For
colonyformationassay,atleast50coloniesthatarebiggerthan100µmfrom4randomelychosen
fieldswereblindlycounted.
1.b.Foranimalstudies,includeastatementaboutsamplesizeestimateevenifnostatisticalmethodswereused.
For Figure 1G, six mice for each category were used for the experiments. For
100,000 GSCs, five mice were used finally. For Figure 8, four groups of mice have
subcutaneously and orthotopically injections to establish tumors, respectively. For
500,000/mouse GSCs, forty mice were used finally. They are followed for a fixed
amount of time and then are sacrificed.
2.Describeinclusion/exclusioncriteriaifsamplesoranimalswereexcludedfromtheanalysis.Werethecriteriapreestablished?
Forcellculturestudies,sampleswerenotexcluded.
3.Wereanystepstakentominimizetheeffectsofsubjectivebiaswhenallocatinganimals/samplestotreatment(e.g.
randomizationprocedure)?Ifyes,pleasedescribe.
Inanimalexperiments(Figure8),micewererandomizedintofourgroupsafterafewweeks,when
tumorvolumehadreachedapproximately180mm3.Forcellculturestudies,cellsfromthesame
frozenstockbatchesculturedinlargedishes,anddistributedtosmalldishesandrandomly
allocatedforthetreatment.
Foranimalstudies,includeastatementaboutrandomizationevenifnorandomizationwasused.
Inanimalexperiments(Figure8),micewererandomizedintofourgroupsafterafewweeks,when
tumorvolumehadreachedapproximately180mm3.
4.a.Wereanystepstakentominimizetheeffectsofsubjectivebiasduringgroupallocationor/andwhenassessingresults Forcolonyformationassay,Coloniesbiggerthan100µmindiameterwereblind-countedfrom
(e.g.blindingoftheinvestigator)?Ifyespleasedescribe.
fourmicroscopicfields.Threeindependentexperimentswereperformed.Forlivecellcounting,at
least50cellsfromrandomlychosenfieldswereblindlycounted.Threeindependentexperiments
wereperformed.
4.b.Foranimalstudies,includeastatementaboutblindingevenifnoblindingwasdone
N/A
5.Foreveryfigure,arestatisticaltestsjustifiedasappropriate?
Yes
Dothedatameettheassumptionsofthetests(e.g.,normaldistribution)?Describeanymethodsusedtoassessit.
Yes.Wespecifiedthetestusedtocalculatep--‐values in the respective figure
legends（page 24, 25, 27, 29). One week following implantation, animals received
50 mg/kg D-luciferin, and tumor implantation was verified by luciferase
bioluminescence.
Isthereanestimateofvariationwithineachgroupofdata?
Meanstandarddeviationwerecalculatedfromthreeindependentexperiments.Anestimateof
variationforeachfactor(groups)wasalsocalculatedbythemultiplereplicate2-factorANOVA.
Isthevariancesimilarbetweenthegroupsthatarebeingstatisticallycompared?
Yes
C-Reagents
6.Toshowthatantibodieswereprofiledforuseinthesystemunderstudy(assayandspecies),provideacitation,catalog PARP1:PLA0184(sigma),Ubiquitin:SAB4503053(sigma),NFκBp65:SAB1305640(sigma),
numberand/orclonenumber,supplementaryinformationorreferencetoanantibodyvalidationprofile.e.g.,
NFκBp50:sc-1190(sigma),IкB-α:10505(sigma),IкB-β:PRS4243(sigma),IкB-ε:10632(sigma),
Antibodypedia(seelinklistattopright),1DegreeBio(seelinklistattopright).
Actin:A-5441(sigma),Nestin:AB5922(Merck),Musashi:Ab-5977(Merck),SOX2:AF2018(R&D
Systems),GFAP:Z0334(Dakocytomation),βIII-tubulin:MMS-435P(Covance),caspase3:9662
(CellSignalingTechnology;CST),IKK-α:2682(CST),IKK-β:2684(CST),JNK:9252(CST),p-JNK:
9251(CST),p38:9212(CST),p-p38:9211(CST),USP14:A300-919(BethylLaboratory),19SRPT5:
BML-PW8770(ENZOLife),20Sα3:BML-PW8115(ENZOLife),20Sα7:BML-PW8110(ENZOLife),
RNF123:Ab57549(Abcam),UBR2:NBP1-45243(NovusBiologicals)
7.Identifythesourceofcelllinesandreportiftheywererecentlyauthenticated(e.g.,bySTRprofiling)andtestedfor
mycoplasmacontamination.
GliomaStemcelllinesareobtainedfromUniversityofPittsburgh.Weconfirmedthatmycoplasma
contaminationusingPlasmocinreagents(InvivoGen).Thereferencesarecitedinthemaintext.
*forallhyperlinks,pleaseseethetableatthetoprightofthedocument
D-AnimalModels
8.Reportspecies,strain,gender,ageofanimalsandgeneticmodificationstatuswhereapplicable.Pleasedetailhousing
andhusbandryconditionsandthesourceofanimals.
Five-week-oldfemaleBALB/cnudemicewereobtainedfromtheShizuokaLaboratoryAnimal
Center(Shizuoka,Japan)andhousedinaspecificpathogen-freeenvironment.Animalswerekept
inIVCcagesinatemperature-controlledenvironmentwitha12hoursdark/lightcycleandfedwith
standardplantbasedchow.
9.Forexperimentsinvolvinglivevertebrates,includeastatementofcompliancewithethicalregulationsandidentifythe AnimalstudieswereconductedaccordingtotheGuidefortheCareandUseofLaboratoryAnimals
committee(s)approvingtheexperiments.
publishedbytheUSNationalInstitutesofHealth(NIHpublicationno.85-23,revisedin1996)and
theprotocols(12-0304)approvedbytheInstitutionalAnimalCareandUseCommitteeatSeoul
NationalUniversity.
10.WerecommendconsultingtheARRIVEguidelines(seelinklistattopright)(PLoSBiol.8(6),e1000412,2010)toensure Compliancewasconfirmed.
thatotherrelevantaspectsofanimalstudiesareadequatelyreported.Seeauthorguidelines,under‘Reporting
Guidelines’.Seealso:NIH(seelinklistattopright)andMRC(seelinklistattopright)recommendations.Pleaseconfirm
compliance.
E-HumanSubjects
Antibodypedia
1DegreeBio
http://www.equator-network.org/reporting-guidelines/improving-bioscience-research-reporting-the-arrive-guidelines-for-reporting-animal-research/
ARRIVEGuidelines
11.Identifythecommittee(s)approvingthestudyprotocol.
N/A
12.Includeastatementconfirmingthatinformedconsentwasobtainedfromallsubjectsandthattheexperiments
conformedtotheprinciplessetoutintheWMADeclarationofHelsinkiandtheDepartmentofHealthandHuman
ServicesBelmontReport.
N/A
13.Forpublicationofpatientphotos,includeastatementconfirmingthatconsenttopublishwasobtained.
N/A
14.Reportanyrestrictionsontheavailability(and/orontheuse)ofhumandataorsamples.
N/A
15.Reporttheclinicaltrialregistrationnumber(atClinicalTrials.govorequivalent),whereapplicable.
N/A
16.ForphaseIIandIIIrandomizedcontrolledtrials,pleaserefertotheCONSORTflowdiagram(seelinklistattopright)
andsubmittheCONSORTchecklist(seelinklistattopright)withyoursubmission.Seeauthorguidelines,under
‘ReportingGuidelines’.Pleaseconfirmyouhavesubmittedthislist.
N/A
17.Fortumormarkerprognosticstudies,werecommendthatyoufollowtheREMARKreportingguidelines(seelinklistat N/A
topright).Seeauthorguidelines,under‘ReportingGuidelines’.Pleaseconfirmyouhavefollowedtheseguidelines.
F-DataAccessibility
18.Provideaccessioncodesfordepositeddata.Seeauthorguidelines,under‘DataDeposition’.
ThedatainGEOArchivefilesweredepositedintheGeneExpressionOmnibus(GEO)ofNCBI
(http://www.ncbi.nlm.nih.gov/geo/)undertheaccessionnumberofGSE62356.
Datadepositioninapublicrepositoryismandatoryfor:
a.Protein,DNAandRNAsequences
b.Macromolecularstructures
c.Crystallographicdataforsmallmolecules
d.Functionalgenomicsdata
e.Proteomicsandmolecularinteractions
19.Depositionisstronglyrecommendedforanydatasetsthatarecentralandintegraltothestudy;pleaseconsiderthe N/A
journal’sdatapolicy.Ifnostructuredpublicrepositoryexistsforagivendatatype,weencouragetheprovisionof
datasetsinthemanuscriptasaSupplementaryDocument(seeauthorguidelinesunder‘ExpandedView’orin
unstructuredrepositoriessuchasDryad(seelinklistattopright)orFigshare(seelinklistattopright).
20.Accesstohumanclinicalandgenomicdatasetsshouldbeprovidedwithasfewrestrictionsaspossiblewhile
N/A
respectingethicalobligationstothepatientsandrelevantmedicalandlegalissues.Ifpracticallypossibleandcompatible
withtheindividualconsentagreementusedinthestudy,suchdatashouldbedepositedinoneofthemajorpublicaccesscontrolledrepositoriessuchasdbGAP(seelinklistattopright)orEGA(seelinklistattopright).
21.Asfaraspossible,primaryandreferenceddatashouldbeformallycitedinaDataAvailabilitysection.Pleasestate
N/A
whetheryouhaveincludedthissection.
Examples:
PrimaryData
WetmoreKM,DeutschbauerAM,PriceMN,ArkinAP(2012).Comparisonofgeneexpressionandmutantfitnessin
ShewanellaoneidensisMR-1.GeneExpressionOmnibusGSE39462
ReferencedData
HuangJ,BrownAF,LeiM(2012).CrystalstructureoftheTRBDdomainofTERTandtheCR4/5ofTR.ProteinDataBank
4O26
AP-MSanalysisofhumanhistonedeacetylaseinteractionsinCEM-Tcells(2013).PRIDEPXD000208
22.Computationalmodelsthatarecentralandintegraltoastudyshouldbesharedwithoutrestrictionsandprovidedina N/A
machine-readableform.Therelevantaccessionnumbersorlinksshouldbeprovided.Whenpossible,standardized
format(SBML,CellML)shouldbeusedinsteadofscripts(e.g.MATLAB).Authorsarestronglyencouragedtofollowthe
MIRIAMguidelines(seelinklistattopright)anddeposittheirmodelinapublicdatabasesuchasBiomodels(seelinklist
attopright)orJWSOnline(seelinklistattopright).Ifcomputersourcecodeisprovidedwiththepaper,itshouldbe
depositedinapublicrepositoryorincludedinsupplementaryinformation.
G-Dualuseresearchofconcern
23.Couldyourstudyfallunderdualuseresearchrestrictions?Pleasecheckbiosecuritydocuments(seelinklistattop
right)andlistofselectagentsandtoxins(APHIS/CDC)(seelinklistattopright).Accordingtoourbiosecurityguidelines,
provideastatementonlyifitcould.
N/A