陳宏山課題組-論文siVIP

Titlepage

TRAP1drivessmoothmusclecellsenescenceandpromotesatherosclerosisviaHDAC3-primedhistoneH4lysine12lactylation

XuesongLi1?,MinghongChen1?,XiangChen1?,XianHe1,XinyuLi1,HuiyuanWei1,YongkangTan1,JiaoMin1,TayyibaAzam2,MengdieXue3,YunjiaZhang1,MengdieDong1,QuanwenYin1,LongbinZheng1,HongJiang1,DaHuo3,XinWang2,ShaoliangChen4*,YongJi5,6*andHongshanChen1,7,8*

1KeyLaboratoryofCardiovascularandCerebrovascularMedicine,SchoolofPharmacy,NanjingMedicalUniversity,Nanjing211166,China.

2FacultyofBiology,MedicineandHealth,UniversityofManchester,Manchester,UK.

3DepartmentofMedicinalChemistry,KeyLaboratoryofCardiovascularandCerebrovascularMedicine,SchoolofPharmacy,NanjingMedicalUniversity,Nanjing211166,China.

4DepartmentofCardiology,NanjingFirstHospital,NanjingMedicalUniversity,Nanjing,China.

5KeyLaboratoryofCardiovascularandCerebrovascularMedicine,KeyLaboratoryofTargetedInterventionofCardiovascularDisease,CollaborativeInnovationCenterforCardiovascularDiseaseTranslationalMedicine,StateKeyLaboratoryofReproductiveMedicine,SchoolofPharmacy,theAffiliatedSuzhouHospitalofNanjingMedicalUniversity,GusuSchool,NanjingMedicalUniversity,Nanjing,Jiangsu,China.

6NationalKeyLaboratoryofFrigidZoneCardiovascularDiseases(NKLFZCD),DepartmentofPharmacology(State-ProvinceKeyLaboratoriesofBiomedicine-PharmaceuticsofChina),CollegeofPharmacy,KeyLaboratoryofCardiovascularMedicineResearchandKeyLaboratoryofMyocardialIschemia,ChineseMinistryofEducation,NHCKeyLaboratoryofCellTransplantation,theCentralLaboratoryoftheFirstAffiliatedHospital,HarbinMedicalUniversity,Harbin,Heilongjiang,China.

7KeyLaboratoryofTargetedInterventionofCardiovascularDisease,CollaborativeInnovationCenterforCardiovascularDiseaseTranslationalMedicine,NanjingMedicalUniversity,Nanjing211166,China.

8DepartmentofCardiovascularSurgery,TheFirstAffiliatedHospitalofNanjingMedicalUniversity,Nanjing,Jiangsu,211166,China.

*Correspondingauthor.Tel:+862586868467,Fax:+862586868467,E-mail:

hongshanchen@

,

yongji@

,

Chmengx@126.com

?Theseauthorscontributedequallytothiswork.

SUPPLEMENTARYFIGURES

SupplementalFigure1.TRAP1regulatesVSMCsenescence.

(A)Sankeydiagramshowingsenescenceandmitochondriongenes.(B)RT-qPCRanalysisofTRAP1mRNAinaortictissuesfromtheNon-ASandASgroups(n=5independentbiologicalreplicates).(C)RT-qPCRanalysisofTrap1mRNAinaortictissuesfromHFD-fedApoeKOmice(n=10independentbiologicalreplicates).(D)RelativemRNAexpressionofTrap1inaorticsamplesfromApoeKOmicefedanHFDfor0,8,12,and16weeks(n=10independentbiologicalreplicates).(E)ImmunofluorescencestainingofTRAP1(red)withtheVSMCmarkerα-SMA(green),endothelialcellmarkerCD31(green),andmacrophagemarkerF4/80(green)intheaorticrootsfromNC-orHFD-fedApoeKOmice(scalebar=50μm,n=6independentbiologicalreplicates).(F)RT-qPCRanalysisofTRAP1mRNAinRas-inducedhVSMCs(n=6independentbiologicalreplicates).(G)WesternblotanalysisofTRAP1inRas-inducedhumanaorticendothelialcells(HAECs)andperipheralbloodmononuclearcells(PBMCs)(n=5independentbiologicalreplicates).(H)RT-qPCRanalysisofTRAP1inRas-inducedHAECsandPBMCs(n=6independentbiologicalreplicates).(I)QuantificationofTRAP1intensityasshowninFigure1E(n=5independentbiologicalreplicates).(J)WesternblotanalysisoftheknockdownefficiencyofTRAP1siRNA(n=5independentbiologicalreplicates).(K)WesternblotanalysisofcontractileandsyntheticphenotypemarkersinRas-inducedhVSMCstransfectedwithsiTRAP1(n=5independentbiologicalreplicates).(L)RT-qPCRanalysisofcontractileandsyntheticphenotypemarkersmRNAsinRas-inducedhVSMCstransfectedwithsiTRAP1(n=6independentbiologicalreplicates).(M)QuantificationofthenumberofSA-β-GalpositivecellsinFigure1H(n=5independentbiologicalreplicates).(N)RelativeSASPmRNAexpressioninRas-inducedhVSMCstransfectedwithsiTRAP1,asshowninFigure1L(n=6biologicalreplicates).(O)WesternblotanalysisofTRAP1overexpressingefficiency(n=5independentbiologicalreplicates).(P)QuantificationofthesenescencemarkerlevelsinFigure1M(n=5independentbiologicalreplicates).(Q)QuantificationofthenumberofSA-β-GalpositivecellsinFigure1N(n=5independentbiologicalreplicates).(R)RelativeSASPmRNAexpressioninhVSMCsoverexpressingTRAP1,asshowninFigure1Q(n=6independentbiologicalreplicates).*p<0.05,**p<0.01,***p<0.001.Dataarepresentedasthemean±SD.Unpairedt-testwasusedforcomparison(SupplementalFigureS1G-I,andS1O-R).Welch’scorrectionwasusedforcomparisonsinS1B-C,andS1F.One-wayANOVAwasperformed(SupplementalFigureS1D,andS1J-N).

SupplementalFigure2.TRAP1isakeyregulatorofenergyreprogramminginsenescentVSMCs.

(A)Super-resolutionfluorescenceimagesshowingtheco-localisationofTRAP1(red)withmitochondria(TOMM20,green)inRas-inducedhVSMCstransfectedwithsiTRAP1(scalebar=10μm,n=5independentbiologicalreplicates).(B)Flowchartofglycolysisinvolvingglycolyticrate-limitingenzymes.(C)WesternblotanalysisofmitochondrialTCAcycle9enzymesinRas-inducedhVSMCstransfectedwithsiTRAP1(n=5independentbiologicalreplicates).(D)Quantificationofproteinlevels(FigureS2C).(E)WesternblotanalysisofelectrontransferchaincomplexesI-VinRas-inducedhVSMCstransfectedwithsiTRAP1(n=5independentbiologicalreplicates).(F)AnalysisoftheactivityofTCAcycleenzymes(n=6independentbiologicalreplicates).(G)Analysisoftheactivityofelectronrespiratorychainenzymes(n=6independentbiologicalreplicates).(H)CellviabilityofRas-inducedhVSMCstreatedwithG-TPP.CellviabilitywasmeasuredusingtheCCK8assay(n=6independentbiologicalreplicates).(I)DetectionofcomplexIVactivityinhVSMCsoverexpressingTRAP1transfectedwithsiPFK1(n=6independentbiologicalreplicates).(J)ProteininteractiondiagramsofTRAP1andPFK1.(K)PFK1immunoblotanalysisofwholelysatesfromTRAP1-deficientsenescenthVSMCsusingananti-ubiquitinantibody(n=5independentbiologicalreplicates).(L)Followingincubatedwith20μMMG132for6h,thehVSMCsweretreatedwithRasandsiTRAP1andcollectedforwesternblotanalysisofthePFK1proteinlevel(n=5independentbiologicalreplicates).(M)Quantificationoftheproteinlevels(FigureS2L).(N)Followingincubatedwith20μMMG132for6h,thehVSMCsweretreatedwithRasandG-TPPandcollectedforwesternblotanalysisofthePFK1proteinlevel(n=5independentbiologicalreplicates).***p<0.001.Dataarepresentedasthemean±SD.One-wayANOVAwasperformed(SupplementalFigureS2H-I,andS2M-N).Unpairedt-testwasusedforcomparison(SupplementalFigureS2D-G).

SupplementalFigure3.TRAP1-mediatedlactateaccumulationpromotesVSMCsenescence.

(A)RelativeSASPmRNAexpressioninRas-inducedsenescenthVSMCstreatedwithsiTRAP1,followedbytreatmentwithorwithoutexogenouslactate,asshowninFigure3E(n=6independentbiologicalreplicates).(B)WesternblotanalysisoftheinterferenceefficiencyofLDHAsiRNA(n=5independentbiologicalreplicates).**p<0.01,***p<0.001.Dataarepresentedasthemean±SD.One-wayANOVAwasperformed(SupplementalFigureS3A-B).

SupplementalFigure4.TRAP1-mediated-H4K12lapromotesSASPactivationinsenescentVSMCs.

(A)Quantificationoftheproteinlevels(Figure4B)(n=5independentbiologicalreplicates).(B)Flowchartoftherun-ontranscriptionanalysis.***p<0.001.Dataarepresentedasthemean±SD.One-wayANOVAwasperformed(SupplementalFigureS4A).

SupplementalFigure5.UpregulationofH4K12laviaHDAC3blockingpromotessenescenceinVSMCs.

(A)QuantificationofHDAC1,HDAC2,HDAC3andp300levelsinFigure5A(n=5independentbiologicalreplicates).(B-D)QuantificationofHDAC3levelsinFigure5B-D(n=5independentbiologicalreplicates).(E)WesternblotanalysisofHDAC3overexpressingefficiency(n=5independentbiologicalreplicates).(F)QuantificationofsenescencemarkersandH4K12lalevelsinFigure5E(n=5independentbiologicalreplicates).(G)QuantificationofH4K12laintensityinFigure5F(n=5independentbiologicalreplicates).(H)QuantificationofH4K12laintensityinFigure5G(n=5independentbiologicalreplicates).(I)RelativeSASPmRNAexpressionshowninFigure5J.(J)ChIPdetectionofH3,NA,andH4K5labindingtotheSASPpromoterregioninRas-inducedhVSMCswithHDAC3overexpression(n=6independentbiologicalreplicates).*p<0.05,**p<0.01,***p<0.001.Dataarepresentedasthemean±SD.Unpairedt-testwasusedforcomparison(SupplementalFigureS5A,S5B,andS5E).One-wayANOVAwasperformed(SupplementalFigureS5C,S5D,S5F,S5G,andS5H-J).

SupplementalFigure6.SMC-specificTrap1knockoutamelioratesatherosclerosis.

(A)WesternblotanalysisofTRAP1levelsinsmoothmusclecells(SMCs),endothelialcells(ECs),andbonemarrow-derivedmacrophages(BMDMs),fibroblasts,mastcells,neutrophils,plasmacells,andTcellsfromApoeKOTrap1SMCKOmice(n=6independentbiologicalreplicates).(B)RT-qPCRanalysisoftherelativeTrap1expressionintheSMCs,ECs,BMDMs,fibroblasts,mastcells,neutrophils,plasmacells,andTcellsfromApoeKOTrap1SMCKOmice(n=10independentbiologicalreplicates).(C)WesternblotanalysisofcontractileandsyntheticphenotypemarkersinMOVASisolatedfromApoeKOTrap1SMCKOandApoeKOTrap1WTmicefedwithanNCorHFD(n=6independentbiologicalreplicates).(D)RT-qPCRanalysisofcontractileandsyntheticphenotypemarkersmRNAsinMOVASisolatedfromApoeKOTrap1SMCKOandApoeKOTrap1WTmicefedwithanNCorHFD(n=10independentbiologicalreplicates).**p<0.01,***p<0.001.Dataarepresentedasthemean±SD.Unpairedt-testwasusedforcomparison(SupplementalFigureS6A-B).One-wayANOVAwasperformed(SupplementalFigureS6C-D).

SupplementalFigure7.DegradationofTRAP1viaPROTACasanovelstrategyforatherosclerosistreatment.

(A)StructureofthePROTACBP3andthemechanismofBP3-mediatedproteindegradation.(B)CellviabilityofBP3-treatedhVSMCs,measuredusingtheCCK8assay(n=6independentbiologicalreplicates).Dataarepresentedasthemean±SD.One-wayANOVAwasperformed(FigureS7B).

SUPPLEMENTARYTABLES

SupplementaryTableS1.VisualizationofdetaileddatafromheatmapsforFigure4F.

Variable

Control

Ras

siTRAP1

siTRAP1+Ras

pConVSRas

pRasVSsiTRAP1+Ras

ICAM1

H3K27ac

0.25±0.03

0.73±0.08

0.16±0.06

0.37±0.06

p<0.0001

p<0.0001

H3K4me1

0.26±0.03

0.71±0.07

0.14±0.05

0.27±0.05

p<0.0001

p<0.0001

H4K12la

0.25±0.02

0.83±0.07

0.12±0.03

0.29±0.07

p<0.0001

p<0.0001

H4K5la

0.44±0.04

0.38±0.03

0.34±0.08

0.46±0.07

0.1101

0.0440

H3

7.94±0.66

8.4±0.56

7.57±0.66

8.37±0.50

0.2198

0.9239

TNF-α

H3K27ac

0.22±0.05

0.7±0.11

0.13±0.03

0.35±0.09

p<0.0001

p<0.0001

H3K4me1

0.14±0.03

0.68±0.06

0.07±0.02

0.29±0.05

p<0.0001

p<0.0001

H4K12la

0.13±0.03

0.77±0.07

0.1±0.02

0.2±0.03

p<0.0001

p<0.0001

H4K5la

0.53±0.14

0.46±0.11

0.36±0.11

0.45±0.07

0.3259

0.8763

H3

8.42±0.94

8.66±0.69

8.55±0.71

8.15±0.73

0.6132

0.2412

IL6

H3K27ac

0.23±0.03

0.85±0.12

0.23±0.04

0.35±0.06

p<0.0001

p<0.0001

H3K4me1

0.14±0.02

0.89±0.15

0.17±0.04

0.29±0.07

p<0.0001

p<0.0001

H4K12la

0.22±0.04

0.79±0.08

0.14±0.07

0.35±0.10

p<0.0001

p<0.0001

H4K5la

0.35±0.07

0.44±0.06

0.44±0.09

0.39±0.04

0.0312

0.0735

H3

7.51±0.54

7.78±0.64

8.19±0.67

7.74±0.68

0.4546

0.9182

IL-1β

H3K27ac

0.2±0.05

0.8±0.08

0.17±0.02

0.37±0.13

p<0.0001

0.0001

H3K4me1

0.19±0.03

0.55±0.10

0.09±0.02

0.32±0.03

p<0.0001

0.0003

H4K12la

0.17±0.03

0.78±0.10

0.11±0.03

0.28±0.07

p<0.0001

p<0.0001

H4K5la

0.35±0.05

0.31±0.07

0.33±0.05

0.4±0.06

0.3281

0.0382

H3

7.31±0.80

8.2±0.94

7.68±1.36

8.23±0.70

0.1086

0.9454

CCL2

H3K27ac

0.25±0.02

0.68±0.13

0.26±0.03

0.3±0.06

p<0.0001

0.0001

H3K4me1

0.16±0.04

0.85±0.16

0.25±0.06

0.22±0.02

p<0.0001

p<0.0001

H4K12la

0.12±0.06

0.5±0.26

0.08±0.04

0.12±0.08

0.0062

0.0076

H4K5la

0.39±0.07

0.38±0.06

0.44±0.08

0.39±0.07

0.7014

0.8157

H3

6.18±1.43

6.07±1.41

5.83±1.10

5.93±1.57

0.8957

0.8781

IL8

H3K27ac

0.13±0.04

0.76±0.17

0.17±0.06

0.27±0.05

p<0.0001

p<0.0001

H3K4me1

0.12±0.03

0.54±0.08

0.16±0.03

0.25±0.05

p<0.0001

p<0.0001

H4K12la

0.1±0.02

0.54±0.11

0.1±0.02

0.25±0.05

p<0.0001

0.0001

H4K5la

0.34±0.05

0.34±0.04

0.3±0.07

0.25±0.07

0.9449

0.0259

H3

7.18±0.55

7.1±0.50

6.95±0.69

7.18±0.33

0.7969

0.7299

Allvaluesaremean±SD(n=6independentbiologicalreplicates).p<0.05wereregardedasstatisticallysignificant.

SupplementaryTableS2.ThemetabolicparametersofApoeKOTrap1WTandApoeKOTrap1SMCKOmicefedwithNCorHFD.

Variable

NC+

ApoeKOTrap1WT

HFD+

ApoeKOTrap1WT

NC+

ApoeKOTrap1SMCKO

HFD+

ApoeKOTrap1SMCKO

pValue

Bodyweight,g

27.5±0.5

34.4±0.8

27.3±1.1

34.1±0.7

0.61

Heartrate/min

641.5±12.2

641.7±14.7

641.0±13.8

638.8±8.1

0.69

SystolicBP,mmHg

112.2±2.7

124.9±2.4

109.7±3.7

124.8±5.8

0.98

DiastolicBP,mmHg

83.1±5.9

97.6±5.8

77.8±4.7

97.5±3.9

0.97

Triglyceride,mmol/L

1.8±0.3

3.8±0.9

2.1±0.4

4.0±0.7

0.72

Totalcholesterol,

mmol/L

10.1±2.0

18.8±1.3

10.7±1.1

17.9±3.1

0.53

HDL-C,mmol/L

1.2±0.3

0.6±0.1

1.2±0.2

0.6±0.1

0.62

LDL-C,mmol/L

2.1±0.2

5.1±0.4

2.2±0.3

5.1±0.3

0.81

Allvaluesaremean±SD(n=6micepergroup).BP,bloodpressure;HDL-C,high-densitylipoproteincholesterolandLDL-C,low-densitylipoproteincholesterol.p<0.05wereregardedasstatisticallysignificant.HFD+ApoeKOTrap1WTvsHFD+ApoeKOTrap1SMCKO,p>0.05.

SupplementaryTableS3.ThemetabolicparametersofApoeKOmicefedwithHFD+VehicleorHFD+G-TPP.

Variable

HFD+Vehicle

HFD+G-TPP

pValue

Bodyweight,g

33.43±0.55

33.38±0.68

0.89

Heartrate/min

658.67±14.22

656±13.25

0.74

SystolicBP,mmHg

121.1±4.74

122.1±4.34

0.71

DiastolicBP,mmHg

94.87±7.24

96.13±7.24

0.77

Triglyceride,mmol/L

2.61±0.24

2.54±0.18

0.58

Totalcholesterol,mmol/L

18.93±2.55

18.19±1.71

0.57

HDL-C,mmol/L

0.69±0.04

0.69±0.09

0.84

LDL-C,mmol/L

5.81±0.87

5.64±0.71

0.73

Allvaluesaremean±SD(n=6micepergroup).BP,bloodpressure;HDL-C,high-densitylipoproteincholesterol,andLDL-C,low-densitylipoproteincholesterol.p<0.05wereregardedasstatisticallysignificant.HFD+Vehicle+ApoeKOvsHFD+G-TPP+ApoeKO,p>0.05.

SupplementaryTableS4.ThemetabolicparametersofApoeKOmicefedwithHFD+VehicleorHFD+BP3.

Variable

HFD+Vehicle

HFD+BP3

pValue

Bodyweight,g

33.4±0.55

33.55±2.00

0.86

Heartrate/min

632.83±10.48

630.5±12.32

0.73

SystolicBP,mmHg

120.93±5.18

121.92±4.05

0.72

DiastolicBP,mmHg

97.62±3.40

98.28±4.19

0.77

Triglyceride,mmol/L

2.73±0.71

2.89±1.01

0.71

Totalcholesterol,mmol/L

16.88±1.96

17.39±1.57

0.63

HDL-C,mmol/L

0.62±0.12

0.61±0.60

0.81

LDL-C,mmol/L

6.52±1.15

6.38±0.88

0.82

Allvaluesaremean±SD(n=6micepergroup).BP,bloodpressure;HDL-C,high-densitylipoproteincholesterol,andLDL-C,low-densitylipoproteincholesterol.p<0.05wereregardedasstatisticallysignificant.HFD+Vehicle+ApoeKOvsHFD+BP3+ApoeKO,p>0.05.

SupplementaryTableS5.Baselinedataforpatientsusedinthisstudy

Non-AS

AS

Age

66

71

73

71

66

71

61

61

74

67

Sex

Men

Men

Men

Men

Men

Men

Men

Men

Men

Men

SystollcBP,mmHg

132

114

130

127

120

155

149

140

142

145

DiastolicBP,mmHg

81

83

77

80

81

91

92

94

104

91

Totalcholesterol(mmol/L)

2.2

2.72

2.79

2.57

2.74

4.49

3.14

4.25

3.43

4.06

LDLcholesterol(mmol/L)

2.25

2.32

2.19

2.16

1.91

1.8

1.84

2.32

2.12

2.04

HDLcholesterol(mmol/L)

1.57

1.36

1.54

1.67

1.31

1.18

2.14

1.77

2.22

1.95

Triglycerides(mmol/L)

1.85

1.87

1.52

1.5

1.06

0.88

0.85

1.47

1.17

1.92

MedicationUsage

Statinuse

NO

NO

NO

NO

NO

Yes

Yes

Yes

Yes

Yes

Aspirin

NO

NO

NO

NO

NO

Yes

Yes

Yes

Yes

Yes

ACEinhibitor/ARB

NO

NO

NO

NO

NO

Yes

Yes

Yes

Yes

Yes

β-blocker

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

BP,bloodpressure;HDLcholesterol,high-densitylipoproteincholesterol;LDLcholesterol,low-densitylipoproteincholesterol;ARB,Angiotensinreceptorblocker.

TableS6.ThesequenceofsiRNAsusedinthisstudy.

Genename

No.

Forwardsequence

Reversesequence

TRAP1

#1

GCUACACCCUGCACUAUAAGA

UUAUAGUGCAGGGUGUAGCGG

#2

GCUGCUGGUGGAUCAGAUAUA

UAUCUGAUCCACCAGCAGCUG

#3

GAUGCUGAGAAGUAUGCAAAG

UUGCAUACUUCUCAGCAUCUU

LDHA

#1

CUGGCAAAGACUAUAAUGUTT

ACAUUAUAGUCUUUGCCAGTT

#2

GCCAUCAGUAUCUUAAUGATT

UCAUUAAGAUACUGAUGGCTT

#3

GUGCUUAUGAGGUGAUCAATT

UUGAUCACCUCAUAAGCACTT

TableS7.RT-qPCRprimerswereusedinthisstudy.

FactororPTM

Forwardsequence

Reversesequence

Human

IL-6

CCTCCAGAACAGATTTGAGAGTAGT

GGGTCAGGGGTGGTTATTGC

IL-8

GACATACTCCAAACCTTTCCACCC

TTCAAAAACTTCTCCACAACCCTC

IL-1β

TTGCCAGCCAGTGACACAAT

GAGAAGGTGGTTGTCTGGGAAT

ICAM-1

AGGTTGAACCCCACAGTCAC

TCTGAGACCTCTGGCTTCGT

CCL2

GATCTCAGTGCAGAGGCTCG

TCTGGGGAAAGCTAGGGGAA

TNF-α

AGGACACCATGAGCACTGAAAGC

AAGGAGAAGAGGCTGAGGAACAAG

MSRA

TGGTTTTGCAGGAGGCTATAC

GTAGATGGCCGAGCGGTACT

HSP60

GTTGGGGGACCGCTCATT

CCCGGCCATCCTTATAGACG

HSPA9

TGGAATGCCGGCCAAGCGAC

GCCTCAACCCAGGCATCACCA

SIRT3

AGCCCTCTTCATGTTCCGAAGTGT

TCATGTCAACACCTGCAGTCCCTT

TRAP1

GTCGCGCAGGCTCACGACAA

CGCAGCCACTTGGGCAGGAT

RECQL4

GCGCTCTACCGGGAATACC

CAGCCCGATTCAGATGGGG

UCP3

TACCCAACCTTGGCTAGACG

GTCCGAGGAGAGAGCTTGC

UCP2

CCCCGAAGCCTCTACAATGG

CTGAGCTTGGAATCGGACCTT

SOD1

GGTCCTCACTTTAATCCTCTATCCAG

CCAACATGCCTCTCTTCATCC

AIFM1

TCTGGACACTGGCAAACATC

GCTTTCCCCAGAAAGACACA

ALDH2

TTCCACAGGGGAGGTCATCT

CCGTTCAATGAGATCCGCCA

ALDH9A1

AGAGCCAACGACACCACTT

TCTGCCGAATCCTGACTTCTT

APEX1

TCTTGGAATGTGGATGGGCT

ACTGTACCCTTCCTTGTCCG

BAK1

CAGGGACAAGTAAAGGCTAC

ACGGGATCAGCCTGC

BCL2

CTTCGCCGAGATGTCCAG

GGCTCAGATAGGCACCCA

BCL2A1

TACAGGCTGGCTCAGGACTAT

CGCAACATTTTGTAGCACTCTG

CHCHD2

GTGGAGGAAGTAATGCTGAGCC

CACAGAGCTTGATGTCACCCTG

COQ7

AATATGGAGCAAACCGCATC

TCCACAAGGGCATCAGAACT

CYCS

GGCGGCTGTGTAAGAGTATCCA

AACCTTACCCCAGTGGTGCTC

DLAT

GCAGGACTCATCACACCTATTGT

GTAGTTTACCCTCTCTTGCTTTGG

GPX1

CAGTCGGTGTATGCCTTCTCG

GAGGGACGCCACATTCTCG

BCL2L1

GATCCCCATGGCAGCAGTAAAGCAAG

CCCCATCCCGGAAGAGTTCATTCACT

GPX4

AAGAGATCAAAGAGTTCGCC

GGTGTCCAAACTTGGTGAAG

GSR

AGGCTTCCTGCTGCTT

GTACACATCCAACATTCACG

SDHC

TTTAGCCCTCAGCTCTGTAT

CACAGTAAGAACCAGGACAA

POLG

GCACAGAGTCAGAAATGTTCA

TCTTCAAACAGCCACTTCA

PYCR1

ATGGACATTTGGGGAAAGG

CTATTCCACCCACAGTAACC

BAX

CCCACCAGCTCTGAACAGTTC

CCAGCCACAAAGATGGTCAC

CAT

GAAGCTATTAGAGTCGGAGGT

GTTTGGCCCAATACGGT

HTRA2

CTCCCCGGAGTCAGTACAACT

AGGATCTCGATATAGACCACGG

PRKACA

ACTGTTCAACTCCCTTTGTC

TGCCTTGTATCCATTGTCTC

POLB

TGAGCCAAGCTATCCACAAGTA

CTACAAACTTCCTTGCAGCAGA

SOD2

GACAAACCTCAGCCCTAACG

GAAACCAAGCCAACCCCAAC

UCP1

ACGACACGGTCCAGGAGTTCC

GTTGCCCAATGAATACTGCC

MT-CO1

CGCCACACTCCACGGAAGCA

CGGGGCATTCCGGATAGGCC

P53

ATGGAGGAGCCGCAGTCAGA

GGCATTCTGGGAGCTTCATC

P16

GAATAGTTACGGTCGGAGG

TTACTGCCTCTGGTGCC

P21

CCGGCGAGGCCGGGATGAG

CTTCCTCTTGGAGAAGATC

MYOCD

ACGGATGCTTTTGCCTTTGAA

AACCTGTCGAAGGGGTATCTG

α-SMA

AAAAGACAGCTACGTGGGTGA

GCCATGTTCTATCGGGTACTTC

CNN1

CTGTCAGCCGAGGTTAAGAAC

GAGGCCGTCCATGAAGTTGTT

MYH11

CGCCAAGAGACTCGTCTGG

TCTTTCCCAACCGTGACCTTC

TAGLN

AGTGCAGTCCAAAATCGAGAAG

CTTGCTCAGAATCACGCCAT

MMP2

TACAGGATCATTGGCTACACACC

GGTCACATCGCTCCAGACT

OPN

CTCCATTGACTCGAACGACTC

CAGGTCTGCGAAACTTCTTAGAT

Mice

IL-6

TGATGGATGCTACCAAACTGGA

GGAGAGCATTGGAAATTGGGG

IL-1β

TAATGCTTGAGCCCACCCTG

GGGGGAGGCTTCTCTACTGA

ICAM-1

AAACCAGACCCTGGAACTGC

CCCATGGGAGCTAAAGGCAT

CCL2

GGCTCAGCCAGATGCAGTTA

TCATTTGGTTCCGATCCAGGTT

TNF-α

CCTCTCATGCACCACCATCA

GCATTGCACCTCAGGGAAGA

IL-8

TTCGAGACCATTTACTGCAA

TCAGGTCTCCCAAATGAAAG

Myocd

AGGAAGTTCCGATCAGTCTTACA

GGTATTAAGCCTTGGTTAGCCAG

α-SMA

CCCAGACATCAGGGAGTAATGG

TCTATCGGATACTTCAGCGTCA

Cnn1

GCACATTTTAACCGAGGTCCT

CTGATGGTCGTATTTCTGGGC

Myh11

ATGAGGTGGTCGTGGAGTTG

GCCTGAGAAGTATCGCTCCC

Tagln

CCAACAAGGGTCCATCCTACG

ATCTGGGCGGCCTACATCA

Mmp2

ACCTGAACACTTTCTATGGCTG

CTTCCGCATGGTCTCGATG

Opn

ATCTCACCATTCGGATGAGTCT

TGTAGGGACGATTGGAGTGAAA

TableS8.ChIPprimerswereusedinthisstudy.

Primersetname

Forwardsequence

Reversesequence

Human

ICAM-1(-1kb)

GTGGATGTCGAGTCTTGGGG

CAACTCGAACCCAGGCTCAT

ICAM-1(p)

GGGGCTAGAGACAGCGATT

CTTGTTGGGTTGGCACAGAG

ICAM-1(+1kb)

CGTGTCCTGTGTGAGTGGG

TTTTCTGGCCACGTCCAGTT

IL-6(-1kb)

CCTTAGAGCCTGGTGTCTGC

TCTGGGGGTTGGAGATGGAT

IL-6(p)

ATAGCCCAGAGCATCCCTCC

GCTACATTTGCCGAAGAGCC

IL-6(+1kb)

CTATCCGGCCCAAGCTTTCT

TGGCCCATTTGGGTTTCTCA

IL-1β(-1kb)

GGCTAGGGTAACAGCACCTG

CTGGGGCAGAGAACATACGG

IL-1β(p)

CCAGCTCTCCTAGCCAATAC

TGAGTGACTTCCCCATGACG

IL-1β(+1kb)

TGTACCTGTCCTGCGTGTTG

CCAGCTTTTCCTAGGGATGGG

CCL2(-1kb)

CCCGGGGTAACTGAGGATTC

TAGGCTCTGGCACAAACCTG

CCL2(p)

AGCATGAAAGTCTCTGCCGC

GAGAAGAAGAGGGGGCCTTAC

CCL2(+1kb)

TGGGAAAACTGAGGCACCAAG

CCATTCTGCACCAAAGGGCT

IL-8(-1kb)

AGGCAACCGTTAGGGAAAAG

GGACACAACCTGGCTTGACT

IL-8(p)

TTGGCTGGCTTATCTTCACC

GGCAGGTGTTAGAACAAGATCA

IL-8(+1kb)

TGAGGTCAAGGGCTAGGAGA

GCACAGCTCTGCCAGCTAC

TableS9.Antibodieswereusedinthisstudy.

FactororPTM

Vendor

CatNumber

TRAP1

Abcam

ab2721

PFK1

SantaCruzBiotechnology

sc-514824

PKM2

Abcam

ab85555

HK2

SantaCruzBiotechnology

sc-374091

LDHA

Proteintech

19987-1-AP

β-actin

Bioworld

AP0060

IgG

Proteintech

30000-0-AP

PanKla

PTMBIO

PTM-1401

H4K12la

PTMBIO

PTM-1411

H4K5la

PTMBIO

PTM-1407

H3K18la

PTMBio

PTM-1406

H3K27ac-ChIPGrade

CellSignalingTechnology

4353

H3K4me1-ChIPGrade

CellSignalingTechnology

5326

H4K8la

PTMBIO

PTM-1405

H4

CellSignalingTechnology

2592

p300

Abcam

ab14984

HDAC1

Abcam

ab109411

HDAC2

Abcam

ab32117

HDAC3

Abcam

ab32369

Hoechst

Beyotime

C1028

MTRed

Invitrogen

M22426

P16

Abcam

ab189034

P21

Abcam

ab109199

P21

CellSignalingTechnology

2536378

P53

Proteintech

10442-1-AP

α-SMA

Proteintech

67735-1-Ig

BrdU

Abcam

ab1893

H3

CellSignalingTechnology

9715

MYOCD

Invitrogen

MA5-24103

Calponin

Proteintech

24855-1-AP

MMP-2

CellSignalingTechnology

87809

Osteopontin

Invitrogen

PA5-94926

GoatAnti-RabbitIgGH&L(HRP)

Abcam

ab6721

GoatAnti-MouseIgGH&L(HRP)

Abcam

ab6789

HRP-conjugatedgoatanti-rabbitsecondaryantibody

JacksonLabs

111035003

HRP-conjugatedgoatanti-mousesecondaryantibody

JacksonLabs

115036003

TableS10.Primersetswereusedinthisstudy.

Products

Primersets

Telomere

GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT

TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA

36B4

CAGCAAGTGGGAAGGTGTAATCC

CCCATTCTATCATCAACGGGTACAA

TableS11.Nuclearrun-onprimerswereusedinthisstudy.

Primersetname

Forwardsequence

Reversesequence

CCL-2

GATCTCAGTGCAGAGGCTCG

TCTGGGGAAAGCTAGGGGAA

ICAM-1

AGGTTGAACCCCACAGTCAC

TCTGAGACCTCTGGCTTCGT

IL-8

GACATACTCCAAACCTTTCCACCC

TTCAAAAACTTCTCCACAACCCTC

IL-6

CCTCCAGAACAGATTTGAGAGTAGT

GGGTCAGGGGTGGTTATTGC

IL-1β

TTGCCAGCCAGTGACACAAT

GAGAAGGTGGTTGTCTGGGAAT

TNF-α

AGGACACCATGAGCACTGAAAGC

AAGGAGAAGAGGCTGAGGAACAAG

Expendedmethods

Humansamples

Atherosclerotic(n=5)andnon-atherosclerotic(n=5)humanaorticsampleswereobtainedfromtheAffiliatedDrumTowerHospitalofNanjingUniversityMedicalSchool.TheclinicalcharacteristicsofthepatientsaresummarisedinSupplementaryTableS5.AllproceduresinvolvinghumansamplesadheredtotheprinciplesoutlinedintheDeclarationofHelsinkiandthestudyprotocolwasapprovedbytheAffiliatedDrumTowerHospitalofNanjingUniversityMedicalSchool(2019-219-01).HumantissuesampleswereusedforwesternblotandRT-qPCRanalyses.

Animals

ApoeKO(B6/JGpt-Apoeem1Cd82/Gpt)andTrap1floxed(Trap1flox/flox)micewerepurchasedfromGemPharmatech(Jiangsu,China).Smoothmusclecell-specificTrap1knockoutmiceonanApoeKObackground(ApoeKOTrap1SMCKO)weregeneratedbycrossingmaleTrap1flox/floxmicewithaTaglnpromoter-drivenCrerecombinaseline(Tagln-Cre).ApoeKOTrap1WTmicewereemployedascontrolanimals.Fortheatheroscleroticmodels,8-week-oldmicewerefedwithanormalchowdiet(NC)orahigh-fatdiet(HFD,20%fatand1.25%cholesterol;XietongOrganism,Shanghai,China)for16weeks.ToassessthetherapeuticeffectoftheTRAP1inhibitorG-TPP(MedBio,China,dissolvedinDMSO)orPROTACofTRAP1(BP3;MedChemExpress,USA;dissolvedinDMSO)ontheprogressionofatherosclerosis,G-TPP(2mg/kg)orBP3(1mg/kg)wereintraperitoneallyinjectedeveryotherdayfor12weeksfollowing4weeksofHFD.Controlmicereceivedintraperitonealinjectionsofthevehicle(0.1%DMSO)

ADDINEN.CITE

ADDINEN.CITE.DATA

1

.Allmiceweregivenadlibitumaccesstowaterandfood,housedinaspecificpathogen-freefacility,andkeptona12hlight/darkcycle.AllanimalproceduresconformedtotheGuidefortheCareandUseofLaboratoryAnimals(NIH)andwereapprovedbytheAnimalCareandUseCommitteeofNanjingMedicalUniversity(IACUC-2304050).Toexcludeoestrogeneffectsonatherosclerosis,weusedonlymalemiceinourexperiments.

Cellculture

Humanvascularsmoothmusclecells(hVSMCs;ScienCellResearchLaboratories,Carlsbad,CA,USA)werepurchasedfromInvitrogen(USA)andculturedinDulbecco’smodifiedEagle’smedium(DMEM,CA,USA)supplementedwith100U/mLpenicillin,100μg/mLstreptomycin,and10%fetalbovineserumaccordingtothemanufacturer’sprotocol.

PrimaryaorticSMCswereisolatedfromApoeKOTrap1WTandApoeKOTrap1SMCKOmiceafter16weeksofHFD.Briefly,cleanedaortaswereharvestedandsubjectedtoenzymaticdigestionin1mg/mLtypeIIcollagenase(Gibco,USA)and0.74U/mLelastase(WorthingtonBiochemical,USA)at37°Cfor10min.Theremainingaortictissuewasfurtherdigestedinanenzymaticsolutionfor1hat37°Cfollowingcarefulremovaloftheadventitiallayer.Afterdigestion,theSMCsweredissociated,washed,andplatedinDMEM/F12medium(Gibco,USA)supplementedwith1%penicillin/streptomycin.Cellsfrompassagesfourtosevenwereusedfortheexperiment.

siRNAandplasmidDNAtransfection

hVSMCsweretransfectedwith100nMsmallinterferingRNA(siRNA)specifictoTRAP1(siTRAP1)orLHDA(siLDHA)orwithscrambled(non-specific)siRNA(Scr)usingLipofectamineRNAiMaxtransfectionreagent(Invitrogen,USA)accordingtothemanufacturer’sprotocol.ThesiRNAsusedinthepresentstudyarelistedintheSupplementaryTableS6.Tooverexpressthetargetedgenes,pcDNA3.1vectorencodingTRAP1(Youbio,China)orHDAC3(Youbio