figure s1dm5migu4zj3pb.cloudfront.net/manuscripts/83000/83185/jci... · 2016. 10. 20. · r6/2 mice...
TRANSCRIPT
5’- -3’23CAGrepeatsT7
PCRamplifica9onofHTT-exon-1,usingaforwardprimerthatincorporatesT7sequence
HTT-e1DNA
5’- -3’ HTT-e1RNA
Transcrip9on T7transcriptase
Incuba9onwithLNA-ASOs
5’- -3’ 5’- -3’
RTwithsequencespecificprimers
5’- -3’ 5’- -3’RT-primer RT-primer
5’- -3’
Superscript-III
Full-lengthHTT-e1cDNA(387nt)
-3’Par9alHTT-e1cDNA(191nt)
191nt
387nt
PCRamplifica9onwithapairofprimersspanningtheCAGrepeat
5’- -3’ -3’
5’-
5’-
Product(387nt) No-product
b cRetrotranscribedcDNAfromaHTT-e1RNA
PCRproductswithprimersspanningtheCAGrepeat
a
Figure S1 relatedtoFigure1andFigure2
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LNA-SCB
LNA-CTGSuperscriptIII +++-
--+--+--
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300400
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LNA-CTG
LNA-SCB
LNA-SCB
LNA-CTG --+---+---
SuperscriptIII +++-
Sanger-sequencingofthePCRproductswithprimersspanningtheCAGrepeatd
Upper 6 TTAGAC - A-CGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGG 63 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 7 TTAGACAACCGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGG 66 Upper 64 GGAGGGAGAGGGGCGGATCCCGGGCCCGCGGTACCGTCGACTGCAGAATTCCGGCTGAGG 123 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 67 GGAGGGAGAGGGGCGGATCCCGGGCCCGCGGTACCGTCGAC 126 Upper 124 AAGCTGAGGAGGCGGCGGCGGCGGCGGCGGCGGTGGTGGCTGTTGCTGCTGCTGCTGCTG 183 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 127 186 Upper 184 CTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGGAAGGACTTGAG 243 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 187 246 Upper 244 GGACTCGAAGGCCTTCATGGATCCCGGGCCCGCGGTACCGTCGACTGCAGAATTCGAAGC 303 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 247 GGATCCCGGGCCCGCGGTACCGTCGACTGCATTATTC T AAGC 306 Upper 304 TTGAGCTCGAGATCTGAGTCCGGTAGCGCTACCTATAGTGAGTCGTATTAATTTCA 359 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | || Lower 307 TTGAGCTCAAGATCTGAGTCCGGTACCGCTACCTATAGTGAGTCGTATTAATTTCA 362
UpperLower
HTT-e1
Figure S1 relatedtoFigure1andFigure2
Figure S1. LNA-CTGs interfere with retrotranscrip9on of HTT-e1. (a) Schema9c representa9on of theexperimentalworkflow.ApureHTT-e1DNAwith23CAGrepeatswastranscribedandtheresultantHTT-e1RNAwasincubatedwithLNA-CTGorLNA-SCB.Ineachcondi9on,HTT-e1RNAwasretrotranscribedwithasequence specific primer. Strong binding of LNA-CTG to CAG repeats impeded full-lengthretrotranscrip9on, resul9ng in a truncated product of 191 nt. Incuba9on with LNA-SCB allowedretrotranscrip9on of a full-length product of 387 nt. PCR was subsequently performed using a pair ofprimers that span the CAG repeat. (b) Gel electrophoresis showing the retrotranscribed cDNA, in eachcondi9on, e.g. in the in the absence (–) or presence (+) of LNA-SCB, LNA-CTG or SuperscriptIII. (c) Gelelectrophoresis showingRT-PCRproducts using a pair of primers that span the CAG repeats; a productcorrespondingtofull-lengthHTT-e1ofabout~380nt(d)wasnotamplifiedinsamplesincubatedwithLNA-CTG.Anaddi9onalproductofabout~200ntthatcorrespondstoafragmentlackingtheCAGrepeatandadjacent nucleo9deswas present (d), sugges9ng that secondary structures containing the CAG repeatsmay be compa9ble with PCR amplifica9on with the set of primers spanning the CAG repeat in theseexperimentalcondi9ons.(d)AlignementoftheSangersequencingoftheupper(~380nt)andlower(~200nt)PCRproductsshowninthefirstlanein(c).TheyellowboxindicatesthemiddlefragmentwithinHTT-e1thatcontainstheCAGrepeat(highlightedinred)thatisnotpresentinthelower(~200nt)PCRproducts.SimilarsequencestothelowerproductweredetectedinthePCRproductsoflanes2and3in(c).
a%ofcellswith
foci
%cells
Numberoffoci≤34-6≥7
LNA-SCB
LNA-CTG
LNA-CT
GLN
A-SCB
Ac9n
HTT-e29-30
WT
Mut
b
HTT-e1*
*
*
*
Figure S2 relatedtoFigure1
c d
Figure S2. RNA foci in HD fibroblasts (68_CAG repeats) transfected with 15 nM of LNA-ASOs. (a)Representa9ve FISH image in HD fibroblasts transfected with LNA-SCB or LNA-CTG using a probecomplementary to theCAG repeat (n=3 independent transfec9ons).NucleiwerevisualizedbyDAPIstaining (right images). Scale bar, 5 µm (b) Representa9ve gel electrophoresis showingHTT RT-PCRproducts from HD fibroblasts transfected with LNA-ASOs (n = 3 independent transfec9ons). (c)Percentageoffibroblastspresen9ngnuclearRNAfociand(d)focidistribu9on,uponexposuretoLNA-CTGorLNA-SCB.Thenumberofcellswithfoci(c)andthenumberoffocipercell(d)wasdeterminedin150-300 cells per random field in LNA-SCB and LNA-CTG transfected cells (n = 3 independenttransfec9ons,in(d)*P<0,001;Chi-Squaretest).
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X1.3.foci.cellX1.3.foci.cell.1 V3 X4.6.foci.cellX4.6.foci.cell.1 V6 X.7.foci.cellX.7.foci.cell.1
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scb replica1
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LNA-SCB LNA-CTG
23xCAG 80xCAG
GFP
Actin
23xCAG-exon1
LNA-CTG LNA-SCB 10 (nM)
CMVp IRES GFP
(CAG)n
Control: 23xCAG_HTT-e1 WT
Mutant: 80xCAG_HTT-e1 Mut
Protein(PolyQ)HTT-exon1expressionvectorsa
LNA-CTG LNA-SCB
80xCAG-exon1
RNAExpression,24hpost-transfec9onb
HTT-e1
Figure S3 relatedtoFigure1
Figure S3, LNA-CTG bind to HTT-e1 CAG repeats in a human neuronal cell context. (a) Schema9crepresenta9on of HTT-e1-GFP expression vectors. HTT-e1 constructs containing 23 CAG repeats(23×CAG)wereusedasthewild-type(unexpanded)HTT-e1,andanHTT-e1constructcontaining80CAGrepeats(80×CAG)wasusedasamodelofmutantHTT.(b)GelelectrophoresisshowingPCRproductsintwo independentexperiments.HTT-e1expressionvectorswereco-transfectedwithLNA-CTGor LNA-SCB (10nM) inneuronal cells.RT-PCRof totalRNAwasperformed,usingprimers that span theCAGrepeatwithinHTT-e1 and primers targe9ngGFP. β-ac9n amplifica9onwas used as an endogenouscontrol.
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LNA-SCB LNA-CTG
80xCAG
_HTTe1/T
ubulin
Tubulin
polyQ
LNA-CTG LNA-SCB
45nM 45nM
HTT-exon1proteinexpressiona
80*CAG_HTT-e1proteinlevels,48hpost-transfec9on
Figure S4 relatedtoFigure1andFigureS3
FigureS4.LNA-CTGsblockthetoxiceffectofmutantHTT-e1withoutaffec9ngHTT-e1proteinlevels.(a)ExpandedHTT-e1(80×CAG_HTTe1)proteinlevels,48haqerco-transfec9onwithLNA-CTGorLNA-SCB(45nM).ValuesobtainedbydensitometricanalysisofWesternblots.An9-polyQdetectstheexpressionof 80×CAG_HTTe1 using an an9body that recognizes glutamines (MAB1574); a representa9veimmunoblot is shown. Data are expressed as a 80×CAG_HTT-e1/α-Tubulin ra9o, and are shown asmean±SEM.ThedatawereanalyzedbyaMann-WhitneyU test (n=5). (b)Lactatedehydrogenase(LDH)celltoxicityassayindifferen9atedSH-SY5Yneuronalcells24hand36haqerco-transfec9onwith80×CAG_HTT-e1expressionvectorandLNA-SCBorLNA-CTG(45nM).Values represent themean foldchangewithrespecttothecontrolnon-transfectedcells±SEM.ThedatawereanalyzedbyaKruskal-WallistestfollowedbyaMann-WhitneyUtestwithBonferronicorrec9onasapost-hoctest(n=5,*P<0.05)
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1.6
2.0
Nontransfected
80XCAGLNA-SCB
80XCAGLNA-CTG
Rela:v
ecelltoxicy(LDH
assay)
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1.2
1.6
2.0
Nontransfected
80XCAGLNA-SCB
80XCAGLNA-CTG
Rela:v
ecelltoxicy(LDH
assay)
*
MutantHTTexon1toxicityb
24hpost-transfec9on 36hpost-transfec9on
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WTLNA-SCBR6/2LNA-SCBR6/2LNA-CTG
Surgery
Bodyweight(g)
b
Age(weeks)
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Latencytofall(secon
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Age(weeks)
Age(weeks)
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Rotarod Rotarod Rotarod Rotarod Rotarod Rotarod Rotarod
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Rotarod Rotarod Rotarod Rotarod Rotarod
HWT HWT HWT HWT HWT
Beforesurgery
APersurgery
* **
Figure S5 relatedtoFigure2
FigureS5.ExperimentaldesignandLNA-CTGeffectsonphenotypicparametersinanR6/2mousemodelofHD.(a)Schemeshowingtheexperimentalprotocolforthebehaviouralanalysisinwild-type(WT)andR6/2miceintrastriatallyinjectedwithLNA-SCBorLNA-CTG.HWT,hangingwiretest.Thebodyweight(b)andlatencytofallinthehangingwiretest(c)areshowngraphicallyfortheWTLNA-SCB,R6/2LNA-SCBandR6/2LNA-CTGmicegroupsatdifferent9mepointsaqersurgery.Resultsareexpressedasmean±SEM(n=7).Bodyweightdatawasanalyzedbytwo-wayANOVAwithBonferronitestasaposthoc.*P<0.05ascomparedwithLNA-SCB–orLNA-CTG–injectedR6/2mice.
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W T L N A -S C B
W T L N A -C T G
Num
bero
ffalls
(60second
s)
Age(weeks)
16rpm 24rpm 32rpm
a
c
FigureS6.Intrastriatalinjec9onofLNA-CTGdoesnotaltermotorbehaviorinWTmicenorthelevelsofseveralstriatalproteins(a)Resultsfromtherotarodtaskperformedat16,24and32rpmfrom8to15weeksofage.Valuesrepresentthenumberoffallswithin60s.(b)Bodyweightatdifferentages,aqerinjec9onofLNA-CTG.In(a)and(b)data,arepresentedasthemean±SEM(WTLNA-SCBn=10;WTLNA-CTGn=9)andanalyzedbytwo-way ANOVA test. (c) Levels of HTT, DARPP-32, ENJ, PHLPP1, CBP, STEP46 and PSD-95 proteins. StriatalhomogenatesofWTmice5weeksaqerinjec9onofLNA-SCBorLNA-CTGwereanalyzedbyWesternblot.Box-plotsrepresentthedensitometricproteinquan9fica9onnormalizedtotubulinandexpressedrela9vetoaLNA-SCB-injectedWTmice.Representa9veimmunoblotsareshown.DatawereanalyzedwithMann-WhitneyUtest(n=7ineachgroups).
Figure S6 relatedtoFigure2andFigure4
WTLNA-SCB
WTLNA-CTG
PHLPP1
Proteinlevels
(%W
T-SCB)
Proteinlevels
(%W
T-SCB)
STEP46CBP
Proteinlevels
(%W
T-SCB)
Proteinlevels
(%W
T-SCB)
PSD-95
DARPP-32
Proteinlevels
(%W
T-SCB)
HTT
Proteinlevels
(%W
T-SCB)
Proteinlevels
(%W
T-SCB)
ENK
HTTTubulin
DARPP-32Tubulin
ENKTubulin
PHLPP1Tubulin
CBPTubulin
STEP46Tubulin
PSD95Tubulin
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Hoechst33258 EM48 Merge
LNA-CT
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Figure S7 relatedtoFigure2
FigureS7.Intrastriatalinjec9onofLNA-CTGdoesnotmodifymHTTdistribu9oninR6/2micestriatum,5weeksaqeradministra9on.NucleiwerestainedwithHoechst,andmHTTwiththeEM48an9body,48haqerLNA-SCBorLNA-CTGadministra9oninR6/2mice.Scalebar,20µm.
Sta
ckin
g
Htt full length
Exon-1
1 2 3 1 2 3 1 2 3 1 2 3
Tubulin
1 2 3 1 2 3 1 2 3 1 2 3
1: WT LNA-SCB
2: R6/2 LNA-SCB
3: R6/2 LNA-CTG
Sta
ckin
g
Htt full length
Exon-1
Tubulin
Figure S8 relatedtoFigure2
a
b
FigureS8.Wild-typeandmutantHTTexpressionlevelsarenotalteredaqerintrastriataladministra9onofLNA-CTG. Representa9ve immunoblots (obtained by incuba9on with MAB5492 an9-HTT an9body (aa1-82)showingwild-typeandexon-1proteinlevelsinthestriatumofR6/2miceat5(a)and(b)11daysaqerLNA-SCBorLNA-CTGadministra9on(n=4percondi9on).
RT-PCRproductsofcor9calsamples,2daysaqeradministra9on
R6/2+LNA-SCB R6/2+LNA-CTG
Ac9n
Gdx
Hu_E1*
Hu_E1
R6/2+LNA-SCB R6/2+LNA-CTG
Hu_E1*
Ac9n
Gdx
RT-PCRproductsofcor9calsamples,10daysaqeradministra9on
Hu_E1
R6/2+LNA-SCB R6/2+LNA-CTG
RT-PCRproductsofcor9calsamples,5weeksaqeradministra9on
Hu_E1*
Ac9n
Gdx
Hu_E1
a
b
c
Figure S9 relatedtoFigure2
Figure S9. LNA-CTGs strongly bind to HTT-e1 CAG repeats in the cortex of R6/2 mice at 2–10 daysfollowing injec9on into the striatum. The gel electrophoresis shows RT-PCR products of the HTT-e1transgene using primers that span the CAG repeat (HTT_E1*) and primers outside the CAG repeat(HTT_E1) at 2 days (a), 10 days (b) and 5 weeks (c) aqer intrastriatal injec9on of LNA-ASOs (n=5).AmplifiedofGdxandβ-ac4nwereusedasinternalcontrols.
b
Mo_ac9n
Hx_e41
HTT_e1*
12345
a
Proteinlevels(%
)
LNA-SCB
LNA-CTG
STHdhQ7/Q7 STHdhQ111/Q111
HTT
Tubulin
LNA-SCB
LNA-CTG
Figure S10 relatedtoFigure2
Figure 10. LNA-CTG transfec9on in mouse striatal cells does not alter wild-type and mutant HTTexpressionlevels.(a)HTTandmHTTproteinlevelsinSTHdhQ7/Q7andSTHdhQ111/Q111cells,respec9vely,24h aqer transfec9onwith25nMLNA-SCBor LNA-CTG.Box-plots representHTTexpression levels (fromdensitometricanalysesofWesternblots)thatwerenormalizedtoα-tubulinandreferredtoHTTlevelsincontrolSTHdhQ7/Q7cells transfectedwithLNA-SCB.DatawereanalyzedwithMann-WhitneyUtestwithBonferronicorrec9on(n=3inSTHdhQ7/Q7cellsandn=4inSTHdhQ111/Q111).Arepresenta9veimmunoblotis shown. (b) Gel electrophoresis showingHTT andβ-ac9n RT-PCR products from STHdhQ111/Q111 cellstransfectedwithLNA-CTGorLNA-SCB(n=5).TheprimersetsHTT_e1*(forHx-e1),Hx_e41(formouseHxexon41)andMo_ac9n(formouseβ-ac9n)wereused(seeTableS2fordetails).
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SCB sCAGs V3 SCB.1 sCAGs.1
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Pif1 Pf4 Pde3b
P2ry12 Med1 Git2
Gpr34 P2ry13 D130043k22Rik
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTGWTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
*
*+
+
* + *+
*
+* *
Figure S11relatedtoFigure3andTableS4
FigureS11.qPCRanalysestovalidatemicroarraytop-deregulatedgenesinthestriatumofR6/2miceinjectedwithLNA-CTGorLNA-SCB.Pif1,Pf4,Pde3b,P2ry12,Med1,Git2,Gpr34,P2ry13andD130043k22RikexpressionlevelswereanalyzedinWTanimalsinjectedwithLNA-SCBandR6/2miceinjectedwitheitherLNA-SCBorLNA-CTG.Rela9vequan9fica9ons(RQ) inthebox-plotsshowexpression levelsreferredtowild-typeanimals.Hprtwas used as a reference gene. Data were analyzed using a linear mixed effects model and Bonferroni’scorrec9onwasappliedformul9plecomparisons(n=5-9pergroup,*P<0.01withrespecttowild-typeLNA-SCBmice;+P<0.01withrespecttoR6/2LNA-SCBmice).
LNA-SCBLNA-CTG
Figure S12relatedtoFigure3
aM
aml2
mR
NA
leve
ls
PrimersspanningtheCAGrepeats,p1 PrimersmappingoutsidetheCAGrepeats,p2
*+
*+
+*
+*
Mef
2a m
RN
A le
vels
N
r3c1
mR
NA
leve
ls
Sat
b1 m
RN
A le
vels
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2 WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
WTR6/2R6/2
LNA-SCBLNA-CTG
CAGrepeat Setofprimersp1:F-p1,R-p1
Setofprimersp2:F-p1,R-p1
F-p1
R-p1
F-p2
R-p2
b
FigureS12.LNA-CTGbindtoseveraltranscriptscontainingCAGrepeats,withoutalteringthecorrespondingproteinlevels. (a) Schema showing the binding site of the primers spanning the CAG repeats (p1) and primersmappingoutsidetheCAGrepeats(p2).(b)RT-qPCRquan9fica9onofofMaml2,Mef2a,Nr3c1andSatb1mRNAinthestriatumofWTanimals injectedwithLNA-SCBandR6/2mice injectedwithLNA-SCBorLNA-CTG,at5dayspost-injec9on.PrimersspanningtheCAGrepeat (leqpanels)andoutsidetheCAGrepeat (rightpanels)wereused.AWTsamplewasusedasareferenceforrela9vequan9fica9on(RQ).Quan9fica9onwasnormalizedtoHprtasareferencegene.PlotsshowmeanRQdistribu9on.DatawereanalyzedusingalinearmixedeffectsmodelandBonferronicorrec9onwasappliedformul9plecomparisons(n=4pergroup,*P<0.01withrespecttoWT-LNA-SCBmice;+P<0.01withrespecttoR6/2-LNA-SCBmice).(c)Protein levels inthestriatumofWT andR6/2miceat2 (upperpanels)and10(lowerpanels)daysaqerLNA-SCBorLNA-CTGintrastriatal injec9on.Box-plotsrepresentthedensitometricproteinquan9fica9onnormalizedtotubulinandexpressedrela9vetoWTmice.DatawereanalyzedbyaKruskal-Wallistest(n=4-5pergroup).Representa9veimmunoblotsareshown.
NR3C1Tubulin
WTLNA-SCB
R6/2LNA-SCB
R6/2LNA-CTG
Proteinlevels(%
WT)
MEF2ATubulin
SATB1Tubulin
MAML2Tubulin
SATB1Tubulin
NR3C1Tubulin
MEF2ATubulin
MAML2Tubulin
c
Figure S12relatedtoFigure3
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150
200
NR3C1 SATB1MEF2AMAML2
NR3C1 SATB1MEF2AMAML2
Proteinlevels(%
WT)
CAGrepeat Setofprimersp1:F-p1,R-p1
Setofprimersp2:F-p1,R-p1
F-p1
R-p1
F-p2
R-p2
a
b
LNA-SCB
*
Figure S13relatedtoFigure3andFigureS6
Figure S13. LNA-CTGs bind to several transcripts containing CAG repeats in WT mice, without altering thecorrespondentexpression levels (a)Schemeshowingthebindingsiteof theprimersspanningtheCAGrepeats(p1) and primers mapping outside the CAG (p2). (b) The expression levels are shown in the striatum ofWTanimals injectedwithLNA-SCBorLNA-CTG,at5weekspost-injec9on.Quan9fica9onwasnormalizedtoHprtasanindependentreferencegene.Box-plotsshowrela9vequan9fica9on(RQ)referredtoaLNA-SCBinjectedmiceanddatawereanalyzedusing linearmixedeffectsmodel (n=7pergroup,*P< 0.001with respectLNA-SCBinjectedmice).
LNA-CTG LNA-SCB LNA-CTG
Satb1,p1 Satb1,p2
LNA-SCB LNA-CTG LNA-SCB LNA-CTG
Mef2a,p1 Mef2a,p2
LNA-SCB LNA-CTG LNA-SCB LNA-CTG
Nr33c1,p1 Nr3c1,p2
LNA-SCB LNA-CTG LNA-SCB LNA-CTG
Arhgap17,p1 Arhgap,p2
LNA-SCB LNA-CTG LNA-SCB LNA-CTG
Maml2,p1 Maml2,p2
LNA-SCB LNA-CTG LNA-SCB LNA-CTG
Crebbp,p1 Crebbp,p2
LNA-SCB LNA-CTG LNA-SCB LNA-CTG
Mdh2,p1 Mdh2,p2
*
*
*
* *
*
RQ
RQ
RQ
RQ
RQ
RQ
RQ
Figure S14, related with Figure 5
0.4
0.6
0.8
1.0
1.2
1.4
WT.LNA.SCB R6.2.LNA.SCB
●
●
●
●
●
●
●
●
●
●
●
●
●
RQ
a
FigureS14.sCAGexpression in the striatumofWTandR6/2mice injectedwith LNA-SCB. (a) sCAGexpressionlevels are shown in totalRNA isolated from the striatumofWTandR6/2animals injectedwith LNA-SCB, at5weeks post-injec9on. Rela9ve quan9fica9on (RQ) represents the densitometric determina9ons of sCAG PCRproductsnormalizedtoU6andreferredtoaWTsamplewithavalueof1.sCAGquan9fica9onisnotshowninLNA-CTGinjectedR6/2mice,sinceLNA-CTGinterferewithRT-PCR.DatawereanalyzedbyaMann-WithneyUtest(n = 4WTmice andn = 9 R6/2mice). (b) sCAGproducts in total RNA and a frac9on enriched in small RNAs.SamplescorrespondtopooledRNAsisolatedfromthestriatumof13WTand11R6/2animalsinjectedwithLNA-SCB,at5weekspost-injec9on.ThepooledsmallRNAfrac9onswereusedinthetransfec9onexperimentsshownin Figure 5. In the RT-PCR amplifica9on we included (CAG)7x and (CTG)7x pure trinucleo9de repeatoligonucleo9desthatwereusedasposi9veandnega9vecontrols,respec9vely.
LNA-SCB LNA-SCBWT R6/2
b
LNA-SCB LNA-SCBWT R6/2
100nt
200nt
C+ C-
5 days post-injection striatum
+
*
*
*
+
Figure S15
FigureS15.LNA-CTGsincreasethelevelsofRn45Sinthestriatumof R6/2mice,5weeksaqeradministra9onofLNA-ASOs.Theexpressionlevelsareshowninthestriatumof WTanimalsinjectedwithLNA-SCBandR6/2miceinjectedwithLNA-SCBorLNA-CTG,at5days(a)or5weeks(b)post-injec9on.Quan9fica9onwasnormalizedtoHprt and/or Tbp as independent reference genes and a WT sample was used as a reference for rela9vequan9fica9on(RQ).DatawereanalyzedusinglinearmixedeffectsmodelandBonferronicorrec9onwasappliedformul9plecomparisons(n=4,miceat5dayspost-injec9on;n=4-9,miceat5weekspost-injec9on;*P<0.05withrespecttoWT-LNA-SCBmice;+P<0.05withrespecttoR6/2-LNA-SCBmice)
b
a
5 weeks post-injection striatum
Hprt,asareferencegene Tbp,asareferencegene
Hprt,asareferencegene
Gene Name Fold change p value adj. P value
A_55_P1980214 -5.85 0.00 0.00Arhgap17 -5.85 0.00 0.00Pif1 -5.60 0.00 0.00Crebbp -5.09 0.00 0.00A_55_P2077546 -4.22 0.00 0.00Trp63 -4.18 0.00 0.00D130043K22Rik -4.08 0.00 0.00Git2 -4.05 0.00 0.00Pf4 -3.10 0.00 0.10Fabp4 -3.00 0.00 0.12Fam48a -2.88 0.00 0.00Mdh2 -2.81 0.00 0.01A230006K03Rik -2.73 0.00 0.00BC051142 -2.69 0.00 0.00chr17:88890039-88905680_F -2.30 0.00 0.00Las1l -2.15 0.00 0.17Gpr137b -2.05 0.00 0.00Mmp27 -1.87 0.00 0.14chr9:30529709-30589042_R -1.83 0.00 0.01Tob1 -1.82 0.00 0.05chr13:112717680-112733330_R -1.81 0.00 0.13NAP017915-001 -1.73 0.00 0.16Gm5124 -1.72 0.00 0.10Angptl4 -1.68 0.00 0.10Ecm1 -1.64 0.00 0.14Tgm2 -1.62 0.00 0.10Gpr137b-ps -1.56 0.00 0.05Tgfbr2 -1.55 0.00 0.12Gadd45b -1.44 0.00 0.18Foxp1 -1.42 0.00 0.10Secisbp2 -1.42 0.00 0.20D11Wsu99e -1.35 0.00 0.11Vwa1 -1.34 0.00 0.12Lpin2 -1.34 0.00 0.01Tmem150c -1.32 0.00 0.10Wfikkn1 -1.30 0.00 0.05Chl1 -1.27 0.00 0.19chr19:59000015-59016865_R -1.26 0.00 0.101700029G01Rik -1.24 0.00 0.16Ier2 -1.19 0.00 0.20Phlpp1 1.22 0.00 0.16Guf1 1.25 0.00 0.20chr4:128727332-128735227_F 1.30 0.00 0.11Ppfia4 1.31 0.00 0.09Pcdhb12 1.38 0.00 0.13P2ry13 1.39 0.00 0.05chr4:105035450-105059250_F 1.39 0.00 0.16chr19:21653968-21671718_F 1.39 0.00 0.02Pde3b 1.43 0.00 0.12Rpl15 1.44 0.00 0.01Med1 1.46 0.00 0.17chr14:61321789-61325711_F 1.46 0.00 0.12Gm10790 1.51 0.00 0.05LOC100861634 1.63 0.00 0.11P2ry12 1.74 0.00 0.05Gpr34 1.74 0.00 0.02
Table S1. Microarray gene expression analysis in the striatum of R6/2 mice injected with LNA-ASOs. Differently expressed genes in LNA-CTG vs LNA-SCB injected mice are shown (Fold Change greater or less than 1.2; adjusted P < 0.2). The dysegulation pattern and the corresponding fold change are indicated for each gene
Target gene/fragment Primer name, Forward (Fw) or Reverse (Rev) Sequence 5'--->3' Annealing T(ºC) Figure
GCAGCAGCAGCAGCAACAGCCGCCACCGCC
Table S2. Primers for qPCR or retrotranscription of human or mouse endogenous HTT, actin, Gdx or exogenously expressed HTT-exon1. Forward (Fw) and Reverse (Rev) primers are shown. The target gene and or fragment are indicated for each pair of primers. Annealing temperature (T) and efficiency for each pair of primers are also shown
Figure 1A, C
Figure 1A,B,C, Figure S1. Figure S2
Figure 1A,B,C; Figure S2
Figure 1B, Figure S2, Figure S3
Human HTT-exon 1, outside the CAG repeat
Human HTT-exon 1, spanning CAG repeat
HTT_e1_Fw
Human HTT-exon 1 with 23_CAG repeats (HTT-e1 expression vector), spanning the CAG repeat
Figure 2D, Figure S9, Figure S10
Human HTT-exon 29-30HTT_e29_Fw
HTT_e30_Rev
TGGATCTTCAGAACAGCACG
TCGACTAAAGCAGGATTTCAGG
Human actinHu_Actin_Fw
Hu_Actin_Rev
CTGGAACGGTGAAGGTGACA
GGGAGAGGACTGGGCCATT
Human HTT-exon 1 transgene, spanning CAG repeat
HTT_e1*_Fw
HTT_e1*_Rev
CGGCTGAGGCAGCAGCGGCTGT
Mouse GdxMo_Gdx_Fw GGCAGCTGATCTCCAAAGTCCTGG
Mouse actinMo_Actin_Fw ACCTTCTACAATGAGCTGCG
Mouse Htt exon 41Htt_e41_Fw
Htt_e41_Rev
Human HTT-exon 1 transgene, outside the CAG repeat
HTT_e1_Fw
Figure 2D, Figure S9Mo_Gdx_Rev
ATGGCGACCCTGGAAAAGFigure 2D, Figure S9, Figure
S10HTT_e1_Rev CTGCTGCTGGAAGGACTTG
AACGTTCGATGTCATCCAGTGTTA
58
62
60
Figure S10CTTGGCATGAGCGAGATTGC
ACTTGTTCCAGTAGGCCGTG
Figure 2D, Figure S9, Figure S10
Mo_Actin_Rev CTGGATGGCTACGTACATGG60
HTT_e1*_Fw ATGGCGACCCTGGAAAAGCTGAT
HTT_e1*_Rev TGAGGCAGCAGCGGCTG
HTT_e1_Rev
GTTCTGCTTTTACCTGCGGC
ATCAGCTTTTCCAGGGTCGC
60
64
60
60
65
60 Figure S1HTT_e1_T7_Rev CCAAAAGACGGCAATATGGT
Human HTT-exon 1 55 Figure S1HTT_e1_T7_Rev-RT CCAAAAGACGGCAATATGGT (specific primer for retrotranscription of HTT-e1)
Human HTT-exon 1 with 23 CAG repeats containing T7 sequence
HTT_e1_T7_Fw GAAATTAATACGACTCACTATAGGTAGCGCTACCGGACTCAGAT
HTT_e1_23CAG_Fw GTCCCTCAAGTCCTTCCAGC 60 Figure S3
HTT_e1_23CAG_Rev GCTGTTGCTGCTGCTGCT
GFP_Fw TGCAGTGCTTCAGCCGCTAC GFP (HTT-e1 expression vector) 60 Figure S3
GFP_Rev TCGCCCTCGAACTTCACCTC
Gene symbol Gene full name Type of primer Sequence 5'--->3' Annealing T(ºC) Efficiency
Forward GCTCTGGTCCCTGTTCACCReverse ACAGAGCTCTCCTGGCACAForward (spanning the CAG repeat) TGTGGGCACTGAGGATGTAAForward (spanning the CAG repeat) ATCCATCGCAAAGTGAGACCForward CTGCTGGAACTGGCTGTGTReverse TGAACATCATGAACCCAGGAForward (spanning the CAG repeat) GGAGCATTGCACTCTGTTCAForward (spanning the CAG repeat) CAATCCCAACCTCCACATTCForward AGGCGAGGACTTTGTCAAGAReverse TCAGAGCCTGCTTTACTGTCAForward (spanning the CAG repeat) GCTTTGTCTTCTCCCTCGTGForward (spanning the CAG repeat) CAAAGTCCTCGCCTTTCTTGForward TGGTGCCTAAGAAATACCACTTGReverse TGAATGTCTGCTGCCTTCTGForward (spanning the CAG repeat) ATCCTTAGCTCCCCCTGGTAForward (spanning the CAG repeat) CGAAAGTCTGTTTCCCCAGAForward CCTGTCCTGCAGTAAGCACAReverse TTCTCCAGCTCCAGGTCACTForward (spanning the CAG repeat) TTCTCTTTTGCCTTCCCAGAForward (spanning the CAG repeat) AGGGAGCCTTGCTGTTGTTForward AGCGTCAAGGACTGTTTGGTReverse AGTGTGGCGGGTTACAGAAGForward (spanning the CAG repeat) TGCCATCTATGAGCAGGAGAForward (spanning the CAG repeat) TCCAGCTGTGCAGACAGAGTForward CCTCACTGGTGGTGAATGTG Reverse TACCATTTTTCCCCCACAAA Forward (spanning the CAG repeat) CACCTCCCCGTGATAGAATG Forward (spanning the CAG repeat) GCCAAGCACAATTGGAGAATForward TGTGGGCACTGAGGATGTAAReverse ATCCATCGCAAAGTGAGACCForward (spanning the CAG repeat) GCTCTGGTCCCTGTTCACCForward (spanning the CAG repeat) ACAGAGCTCTCCTGGCACAForward GGAGCATTGCACTCTGTTCAReverse CAATCCCAACCTCCACATTCForward (spanning the CAG repeat) CTGCTGGAACTGGCTGTGTForward (spanning the CAG repeat) TGAACATCATGAACCCAGGAForward GCTTTGTCTTCTCCCTCGTGReverse CAAAGTCCTCGCCTTTCTTGForward (spanning the CAG repeat) AGGCGAGGACTTTGTCAAGAForward (spanning the CAG repeat) TCAGAGCCTGCTTTACTGTCAForward CGTGAGACGTACGAGATGTTGReverse TGAAGTAGGTGCTGGTGCTGForward GTAGCACTGCCGTGGAAAATReverse TGTGAGCGCTAGGAACTGAAForward TGCTGTTCTCTTTGGTGGTGReverse ACACGTTGCTGTAACCGAGAForward TTCATGGATTTAACAACAAAACAGAReverse CATGTTCACTGACATTTCAACTCAForward CAAGTTCAGATGCTGGGTCAReverse CCTGTGACGATGAGGGCTATForward TTGCTGCCTCTAGTGTTAAGGAReverse GACAATGGATAAGGCCTGGAForward GAGGTTGGGCAGGTAAGGTTReverse TCCTGGCTTCTCTCTTTCCAForward CAGCCCCACAGACAGAATTTReverse TTTCACAACTGGGGCACATAForward GCGATGGAGATCTTAGCTGTGReverse GGCAAATTTTCCTCCCATTCForward TCTTCGTGTGCTGGACTTTGReverse GCCTCTCCCCAGTTAATTCCForward CGCGGTTCTATTTTGTTGGTReverse TCGTCTTCGAAACTCCGACTForward TGTTGTTGGATATGCCCTTGReverse GGACGCAGCAACTGACATTForward TGCTGTTGGTGATTGTTGGTReverse GTCCTGCAATACTGGGAAGG
Nr3c1
Nr3c1
Glucocorticoid Nuclear Receptor Variant 1
Glucocorticoid Nuclear Receptor Variant 1
60
60
Crebbp CREB Binding Protein 60
Maml2 Mastermind-Like Transcriptional Coactivator 2 56 2043
Mef2A Myocyte Enhancer Factor 2A 56
1617
Crebbp CREB Binding Protein 60 1821
Arhgap17 Rho GTPase Activating Protein 17 60 1683
Arhgap17 Rho GTPase Activating Protein 17 60 1856
1963
Satb1 SATB Homeobox 1 60 2035
Trp63 Transformation related protein 63 64º 1.978
Mdh2 Malate Dehydrogenase 2, NAD (Mitochondrial)
1909
Mef2A Myocyte Enhancer Factor 2A 60 1906
Tbp TATA box binding protein 60º 1.894
RIKEN cDNA D130043K22 gene 60º 1.925
Git2 G protein-coupled receptor kinase-interactor 2 60º 1.886
Med1 Mediator complex subunit 1 60º 1.992
60ºP2ry12 Purinergic receptor P2Y, G-protein coupled 12
Rn45S 45S Pre-Ribosomal RNA 60 1976
1.71560º
Table_S3. Selected genes for qPCR validation, and the pair of primers used for each gene. Annealing temperature (T) and efficiency for each pair of primers are shown.
Arhgap17 Rho GTPase activating protein 17 60º 1.683
Crebbp CREB binding protein 60º 1.617
Rho GTPase activating protein 17Arhgap17 60º 1.856
Crebbp CREB binding protein 60º 1.821
P2ry13 Purinergic receptor P2Y, G-protein coupled 13 60º 1.77
Pde3b Phosphodiesterase 3B, cGMP-inhibited 60º 1.934
D130043K22Rik
G protein-coupled receptor 34Gpr34
Hprt Hypoxanthine guanine phosphoribosyl transferase 60º 1.929
Pf4 Platelet factor 4 60º 1.862
Pif1 PIF1 5'-to-3' DNA helicase homolog (S. cerevisiae) 60º 1.741
1.861
Mdh2 Malate dehydrogenase 2, NAD (mitochondrial) 60º 1.933
1.691
Satb1 SATB Homeobox 1 60 1854
Mdh2 Malate dehydrogenase 2, NAD (mitochondrial) 60º
60 1691
Mdh2 Malate Dehydrogenase 2, NAD (Mitochondrial) 60 1933
2021
1521
Maml2 astermind-Like Transcriptional Coactivator 2 60