shFBW7

shControl

Actin

USP9X

FBW7α

A

Flag-FBW7β

shUSP9xMG132

+ + + +

− + − +shControl

USP9X

Flag-FBW7β

Actin

B

Supplementary Figure 1. (A) iBAQ plot showing MS-enrichment of FBW7 and interaction partners over a control IgG. (B) Western blots for indicated proteins in cells transfected with indicated shRNAs. (C) Western blots for indicated proteins in cells co-transfected with Flag-FBW7β and indicated shRNAs.

46

810

log

iBAQ

FBW

7 IP

4 6 8 10log iBAQ control IP

FBW7USP9X C

Flag-c-Jun + +

+ + HA-Ub

GST-USP9X − +

Ubi

-c-J

UN

WB: FlagIP: HA

260

140

100

75

50

40

HA-Ubiquitin & Flag-FBW7overexpression in HCT116

IP: HA/HA-peptideElution

Flag-M2 IP on pooled sequentially eluted HA-ubiquitin complexes

3x wash

Ubi-Crest Assay on beads bound HA-ubiquitylated FBW7

Western blotting for K48 specific linkage

A B

No DUB

USP2OTU

BAIN1

AMSHUSP9X

Ubi

-Fla

g-FB

W7α

α−K48: WB

α-Flag: WB

UM

K48

-Ubi

-FB

W7α

Stripped/reprobed

Supplementary Figure 2. USP9X cleaves K48-linked polyubiquitin chains on FBW7. (A) Recombinant USP9X does not affect polyubiquitination of c-JUN in an in vitro deubiquitylation reaction. (B) Schematic for experiment in C. (C) Ubiquitin chain restriction (UbiCRest) experiment with indicated deubiquitinases: USP2 (promiscuous), OTUBAIN1 (K48-linked), and AMSH (K63-linked).

C

Flag INPUT

USP9X

shU9 #3

shControl

shU9 #4

USP9x

c-Jun

c-Myc

Actin

HCT116

Cyclin E

Supplementary Figure 3. Accumulation of SCF(FBW7) substrates in cells transfected with USP9X-shRNAcompared to a non-targeting control.

A B

Olfm4 ISH

Usp

9xΔ

GU

sp9x

+/+

MCM6

Alkaline phosphatase

C D

Usp

9xΔ

GU

sp9x

+/+

Usp

9xΔ

GU

sp9x

+/+

Usp

9xΔ

GU

sp9x

+/+

Chromogranin

E

0

1.0

2

0.5

Usp9xFbw7

1.5

Rel

ativ

e m

RN

A ex

pres

sion

(

norm

aliz

ed to

Act

in)

Usp9x+/+

Usp9xΔG

*

Usp

9xΔ

GU

sp9x

+/+

BrdU

0

3

6

9

Usp9x+/+

Usp9xΔG

Brd

U+

cells

/col

onic

cry

pt

*

F

Supplementary Figure 4. (A) IHC sections stained for proliferating cells (MCM6) from the intestine of indicated mice. Scale bar = 50 μm. (B) BrdU staining for proliferating cells in colonic crypts from indicated mice, quantification shown in right panel. Scale bar = 100 μm, n = 3–4 mice/group. (C) In situ hybridization for Olfm4 (stem cells) on the sections from the intestine of indicated mice. Scale bar = 100 μm. (D and E) IHC for enterocytes (Alkaline phosphatase) and enteroendocrine cells (Chromogranin) in gut from indicated mice. Scale bars = 100 μm. (F) qRT-PCR analysis showing mRNA levels of indicated genes normalised to actin and represented as fold change over control, in isolated crypts from Usp9x+/+ (wildtype) and Usp9xΔG gut, n = 5–8 mice/group.

0

2

4

6G

oble

t cel

ls/v

illus

c-MycΔG/+

c-JunΔG/+

Usp9xΔG

Usp9x

+/+0

2

4

6

8

10

12

Brd

U+

cells

/cry

pt

Vehicle DBZ

Usp9xΔG

A B

-

Supplementary Figure 5. (A) Average goblet cell number per villus from indicated mice. (B) Average BrdU+ cell number per crypt from indicated mice.

A B170

150

130

110

90

70

501 2 3 4 5 6 7 8 9 10 110

(weeks)

Per

cent

wei

ght

Usp9x+/+

Usp9xΔG

p <0.0001

1 2 3 4 5 6 7 8 9 10 110(weeks)

150

130

110

90

70

50

Fbw7+/+

Fbw7ΔG/+

p <0.05Per

cent

wei

ght

Supplementary Figure 6. (A and B) Weight curves presented as percent of starting weight in indicated mice from colitis-driven tumorigenesis experiment, n = 5–9 animals/genotype. P values were calculated by Log-rank (Mantel-Cox) test.

SupplementaryTable.ShortlistedcandidatesfromIP-massspectrometryexperimentusingendogenousFBW7asbait.

GeneSymbol Description FunctionFBXW7 F-box/WDrepeat-containingprotein7 E3LigaseSKP1 S-phasekinase-associatedprotein1 SCFComponentUBAP2L Ubiquitin-associatedprotein2-like UbiquitylationMYCBP2 MYCbindingprotein2 ProbableE3ubiquitin-proteinligaseHERC2 E3ubiquitin-proteinligaseHERC2 E3LigaseHUWE1 E3ubiquitin-proteinligaseHUWE1 E3LigaseHECTD1 E3ubiquitin-proteinligaseHECTD1 E3LigaseUSP9X Probableubiquitincarboxyl-terminalhydrolaseFAF-X DeubiquitinaseUSP20 Ubiquitincarboxyl-terminalhydrolase20 DeubiquitinaseFKBP8 Peptidyl-prolylcis-transisomeraseFKBP8 ChaperonFKBP4 Peptidyl-prolylcis-transisomeraseFKBP4 ChaperonPSMA2 Proteasomesubunitalphatype-2 ProteasomesubunitPSMA3 Proteasomesubunitalphatype-3 ProteasomesubunitPSMA6 Proteasomesubunitalphatype ProteasomesubunitPSMD3 26Sproteasomenon-ATPaseregulatorysubunit3 ProteasomesubunitFOXP2 ForkheadboxproteinP2 Transcriptionfactorc-JUN TranscriptionfactorAP-1 TranscriptionfactorSTAT1 Signaltransducerandactivatoroftranscription1-alpha/beta TranscriptionfactorMSH6 DNAmismatchrepairproteinMsh6 DNAreplication/GenomeintegrityMCM3 DNAreplicationlicensingfactorMCM3 DNAreplication/GenomeintegrityMCM7 DNAreplicationlicensingfactorMCM7 DNAreplication/GenomeintegrityTOP1 DNAtopoisomerase1 DNAreplication/GenomeintegrityRAD21 Double-strand-breakrepairproteinrad21homolog DNAreplication/GenomeintegrityNAP1L1 Nucleosomeassemblyprotein1-like1 DNAreplication/GenomeintegrityHDAC2 Histonedeacetylase2;Histonedeacetylase DNAreplication/GenomeintegrityCHD4 Chromodomain-helicase-DNA-bindingprotein4 DNAreplication/GenomeintegrityMRE11A Double-strandbreakrepairproteinMRE11A DNAreplication/GenomeintegrityDNAJB6 DnaJhomologsubfamilyBmember6 DNAreplication/GenomeintegrityMTOR Serine/threonine-proteinkinasemTOR KinaseSTK3 Serine/threonine-proteinkinase3 KinasePANK2 Pantothenatekinase2,mitochondrial KinaseGALK1 Galactokinase KinaseDEK ProteinDEK KinaseCSNK1A1 CaseinkinaseIisoformalpha KinasePI4KB Phosphatidylinositol4-kinasebeta KinaseWEE1 Wee1-likeproteinkinase KinaseXPO1 Exportin-1 NuclearexportCSE1L Exportin-2 NuclearexportKPNB1 Importinsubunitbeta-1 NuclearimportRANBP1 Ran-specificGTPase-activatingprotein TransportLUC7L PutativeRNA-bindingproteinLuc7-like1 RNAbinding/processing

DDX39 ATP-dependentRNAhelicaseDDX39A RNAbinding/processingMARS Methionine-tRNAligase,cytoplasmic RNAbinding/processingRAE1 mRNAexportfactor RNAbinding/processingBCAS2 Pre-mRNA-splicingfactorSPF27 RNAbinding/processingLARS Leucine--tRNAligase,cytoplasmic RNAbinding/processingRBM39 RNA-bindingprotein39 RNAbinding/processingRBM28 RNA-bindingprotein28 RNAbinding/processingDDX24 ATP-dependentRNAhelicaseDDX24 RNAbinding/processingTARS Threonine--tRNAligase,cytoplasmic RNAbinding/processingFMR1 FragileXmentalretardationprotein1 RNAbinding/processingIARS Isoleucine--tRNAligase,cytoplasmic RNAbinding/processingYBX2 DNA-bindingproteinA;Y-box-bindingprotein2 RNAbinding/processingMLL Histone-lysineN-methyltransferase EnzymeRPN2 DPDglycosyltransferasesubunit2 EnzymeDPM1 Dolichol-phosphatemannosyltransferase EnzymePRMT1 ProteinarginineN-methyltransferase1 EnzymeACLY ATP-citratesynthase EnzymeEIF4G1 Eukaryotictranslationinitiationfactor4gamma1 TranslationEIF3F Eukaryotictranslationinitiationfactor3subunitF TranslationEIF2B4 TranslationinitiationfactoreIF-2Bsubunitdelta TranslationEIF5B Eukaryotictranslationinitiationfactor5B TranslationMFF Mitochondrialfissionfactor MitochondrialSSBP1 Single-strandedDNA-bindingprotein,mitochondrial MitochondrialATP5J2 ATPsynthasesubunitf,mitochondrial MitochondrialMTCH2 Mitochondrialcarrierhomolog2 MitochondrialMAGED2 Melanoma-associatedantigenD2 CelladhesionACTR2 Actin-relatedprotein2 CytoskeletonTUBA1C Tubulinalpha-1Cchain CytoskeletonTUBB2A Tubulinbeta-2Achain Cytoskeleton