supplementary information to the manuscript · rossi, agustin f. fernandez, fatima carneiro, carla...

Supplementary Informationto the manuscript

A TRBP Mutation in Human Cancer Impairs microRNA Processing and DICER1 Function

Sonia A. Melo, Santiago Ropero, Catia Moutinho, Lauri A. Aaltonen, Hiroyuki Yamamoto, George A. Calin, Simona Rossi, Agustin F. Fernandez, Fatima Carneiro, Carla Oliveira, Bibiana Ferreira, Chang-Gong Liu, Alberto Villanueva, Gabriel Capella, Simo Schwartz Jr, Ramin Shiekhattar & Manel Esteller

Supplementary Table 1. Primer sequences and probes used.

Supplementary Figure 1. FISH analysis of TRBP, TRBP RNA interference and quantification of primarymicroRNA trancritps.

Supplementary Figure 2. HeatMap, qPCR validation of microRNA array and downregulation of targets of overexpressed miRs upon TRBP transfection in Co115 cell line.

Supplementary Figure 3. Transfection of Precursor microRNA molecules 125b1, 125b2 and let-7a.

Supplementary Figure 4. DICER1 studies.

Supplementary Figure 5. Tumor supressor-like features of the TRBP/DICER1 complex.

Supplementary Figure 6. TRBP short harpin experiments, TRBP induced overexpression and DICER1 RNAi.

Supplementary Figure 7. TRBP protein expression in normal colon mucosa and microdissected primary colontumors according to TRBP mutational status.

Nature Genetics: doi:10.1038/ng.317

Supplementary Table 1. Primer sequences and probes used.

Mutation analysis of genomic DNA

Gene Location Repeat Repeat location Primers

AGO1 1p35-p34 7C Exon 2 F:5’TGCCAGACTTTACCCTCACC3’ R:5’TTGATTGGTTTCCCCACAGT3’

AGO1 1p35-p34 6G Exon 5 F:5’GACCAGTTCTCTGCCTGTCC3’ R:5’ATAGCTCTCCCCACTCACCA3’

AGO1 1p35-p34 6C+6A Exon 14 F:5’AGGTACCATGCTGGGAATTG3’ R:5’AATCATGGGCAGTTTTGAGG3’

AGO2 8q24 6C Exon 2 F:5’AATGCAATATTGGCCTGGAC3’ R:5’TGGAGGCTTGAAGGCATATC3’

AGO2 8q24 6A Exon 9 F:5’ACTGACGGCCTGTCTCTACG3’ R:5’GTCGCTCTGATCATGGTTGA3’

AGO2 8q24 6C Exon 11 F:5’TCTCACCCTGAACCTTTTGC3’ R:5’GGGGGCTTACTTCAGATGGA3’

AGO2 8q24 7C Exon 14 F:5’CATCTTTCTGGGAGCAGACG3’ R:5’CCCTACCGCAGCTTAGTGAG3’

AGO4 1p34.3 6A Exon 13 F:5’GGAGGTGAAACGTGTTGGAG3’ R:5’TCAGGCAAAGATTGGAAAGG3’

AGO4 1p34.3 6C Exon 14 F:5’GTTCCAGCAGCCTGTCATCT3’ R:5’GGTCCTGGATGACCTCTTGA3’

AGO4 1p34.3 6A Exon 15 F:5’TTACCGGCCAGGAATAACTT3’ R:5’CTCTTGCCAGAGCCTCAGAT3’

DGCR8 22q11.2 6A Exon 5 F:5’TCGAAGCTACCTGGAGAAGC3’ R:5’TTCTTTGTGGGTGCATCTTG3’

DGCR8 22q11.2 6A Exon 7 F:5’GGTGACCATTGATGGTGTGA3’ R:5’CAGGCAGCACGTATCTTTGA3’

DICER1 14q32.13 6A Exon 21 F:5’TTGCTGTTGCTCTCAGCCTA3’ R:5’AGGGACAATTGTGCTGTGCT3’

DICER1 14q32.13 6A Exon 22 F:5’TTGAATGAATTCCAGCAGTGA3’ R:5’AACCAGGAGGAAGCCAATTC3’

DICER1 14q32.13 6GGA+6A Exon 23 F:5’GCTATGTTTCCCCTCCTTCC3’ R:5’CCCCACCACAAAGTCATCTT3’

DROSHA 5p13.3 6C+6C+6C Exon 4, F:5’CCTCCACGACCAGACTTTGT3’

R:5’AGGTGGGAAGTTGTGGTGAG3’

DROSHA 5p13.3 6A Exon 5 F:5’GAGACACAGGCATCGAGACA3’ R:5’GCTGTTGCTCCATACAAGCA3’

DROSHA 5p13.3 6C Exon 12 F:5’ATTTGGGGTTCGTGTTTCTG3’ R:5’TGGAAAGAAGGGATGAAGGA3’

DROSHA 5p13.3 6A Exon 19 F:5’AATGCTTGATGTTTGGCTTACT3’ R:5’CTCAGAAGTCTCCCCACCTG3’

PACT 2q31.2 6A Exon 6 F:5’CGTTTGATCATCCTCTGTGG3’ R:5’TCTCAGCAGCATTCCTTTTG3’

TRBP 12q12-q13 6G Exon 3 F:5’TTTCCCGTCCTTTCAGTGAC3’ R:5’GGTTCCTTGAAGGGCTTCTG3’

TRBP 12q12-q13 7C Exon 5 F:5’CGGGAGATGGTAGTCAGGAA3’ R:5’AAATGAGGATGGGACACACC3’

TRBP 12q12-q13 6A Exon 7 F:5’ATAACCCAGCAGCCCTCTCT3’ R:5’GTCATCATCAGGCTCCACCT3’


Amplification of primary and precursor microRNA transcripts

Forward 5’ 3’ Reverse 5’ 3’

Pri-miRs

hsa-pri-miR 7-1 AAAACTGCTGCCAAAACCAC GCTGCATTTTACAGCACCAA

hsa-pri-mir-10a CGCGGAAAGTAGGAGAACTG AGGAAGGAGTCTTCGTGTGG

hsa-pri-miR 21 TGTTTTGCCTACCATCGTGA AAGTGCCACCAGACAGAAGG

hsa-pri-miR 22 CATGCCCTGCTCAGATCTTT CAGCCCATTTCTGTCACCTT

hsa-pri-mir-26a-1 CTCTCCCGAGGGAATGAAG ACTCTGGTGTTGGTGCCTCT

hsa-pri-miR-30a TACAGAATCGTTGCCTGCAC TGCTCCTAAAGTAGCCCCTTG

hsa-pri-miR-31 GTGTTTTCCCTCCCTCAGGT GGAAATCCACATCCAAGGAA

hsa-pri-mir-99a TGCATCCTTAGAACTCAGCATTT ATTGTTGAACGGCACTGTGT

hsa-pri-mir-100 ATGTCACAGCCCCAAAAGAG AGCCCCCTTTTCCATTTTTA

hsa-pri-mir-125a TGCCTATCTCCATCTCTGACC TGGTGGTCAAATGTCATGCT

hsa-pri-miR 125b1 CCATACCACCTGTTTGTTGC TCCACCAAATTTCCAGGATG

hsa-pri-mir-125b-2 GGAAGAAGAATTCTACCGCATC GGATGGGTCATGGTGAAAAC

hsa-pri-mir-150 TGGGTATAAGGCAGGGACTG GAGTACAGGGAGGGGAGGTC

hsa-pri-mir-181a-2 CTGGTTCTTGGGATGTGGAT GATTGCAGGACCATTTCTGG

hsa-pri-miR 193b GGGAAAAGAGGCTTTTGGAG TAGCAAACCTCCCCCTCTTT

hsa-pri-mir-194-1 CTCCCATGATGAGCAAAAGG TTTCTGCTGGAATCAAATGAGA

hsa-pri-mir-196a-1 CTTCTGTTGGGGAAGAGGTG CCAGCTAAGCAAGGAGCAGT

hsa-pri-miR-625 TCTGAGGCTTGCAGGTTTTT CAGCCCCATCTATCCTTTGA

hsa-pri-miR-601 GAGTCCAGACCAGAGCCATC TGAGAGCCAGACAGACATGG

hsa-pri-mir-613 GGTGTGGGCTTTATTGGTTG CATGCTGTGGCCTTCCTTAC

hsa-pri-mir-627 GGTGAGGATGAATTGCTGGT CCCCTCATAAGCCAACAAGA

hsa-pri-mir-205 ACAGGCTGAGGTTGACATGC GAGTTACTCTTGCTGCTGCTG

hsa-pri-mir-212 CCCTCTGGGACATCTTTGAC CTGTTACCTCCAGTTCCCACA

hsa-pri-mir-216 AGGGGTACATCAGGGCTTCT GGTTGCCATGCAAGTCTTTT

hsa-pri-mir-375 AGACCAGGACCAGGAGATCA ACCCGTACGGTTGAGATGG

hsa-pri-mir-425 CAGGTCATGCACCTTCAGAAT CCACCCCCATTCCTTTTAAT

hsa-pri-let7a TTTCACCATTCACCCTGGAT CCAGGCCATAAACAAATGCT

hsa-pri-let-7f-1 CCATTCCAGAAGAAAACATTGC TGCCTACTGTACTACTTGAACA

Pre-miRs

hsa-pre-miR 7-1 TGGCCTAGTTCTGTGTGGAA GCAGACTGTGATTTGTTGTCG

hsa-pre-miR-19a CCTCTGTTAGTTTTGCATAGTTGC CAGGCCACCATCAGTTTTG

hsa-pre-miR 21 ATGTTGACTGTTGAATCTCATGG TGTCAGACAGCCCATCGAC

hsa-pre-miR 22 TGGCAAGCTTTATGTCCTGA GCAGAGGGCAACAGTTCTTC

hsa-pre-miR 30a ATCCTCGACTGGAAGCTGTG CTGCAAACATCCGACTGAAA

hsa-pre-miR 31 GAGAGGAGGCAAGATGCTG ATGTTGGCATAGCAGGTTCC

hsa-pre-miR125b1 CAGTCCCTGAGACCCTAACTTG AGAGCCTAACCCGTGGATTT

hsa-pre-miR-138-1 GGGCAGCTGGTGTTGTGA GTGTGGCCCTGGTGTTGT

hsa-pre-miR-148a AGGCAAAGTTCTGAGACACTCC TGCACTGACTTCTATCATACTCAGA

hsa-pre-miR 193b GGTCTCAGAATCGGGGTTTT CTTTGAGGGCCAGTTGGATA

hsa-pre-miR 212 GGCACCTTGGCTCTAGACTG CCGTGACTGGAGACTGTTACTG

hsa-pre-miR let7a

TGTGGGATGAGGTAGTAGGTTG

TATCTCCCAGTGGTGGGTGT


Northern Probes

hsa-let7-a CCCTAACTATACAACCTACTACCTCAAATCCCAGTGTGGG

hsa-miR-193 CCCCAAAAGCGGGACTTTGAGGGCCAGTTGGATAAAACATAAACTCAT

hsa-miR-212 GGGGCCAGGCGTCGGTGGCCGTGACTGGAGACTGTTACTGAGGGCGGCCC

hsa-miR-31 CCCAGTTCAACAGCTATGCCAGCATCTTGCCTCCTCTCC

hsa-miR-143 CTCTCTTCCTGAGCTACAGTGCTTCATCTCAGACTCCCAACTG

hsa-miR-22 GGGCAACAGTTCTTCAACTGGCAGCTTTAGCTGGGTCAGG

hsa-miR-125b CCCGTGGATTTAAACGGTAAACATCACAAGTTAGGGTCTCAGGGACTG

5.8S rRNA GCCCCGGGAGGAACCCGGGGCCGCAAGTGCGTTCGAAGTGTCGATGAT

For TRBP cDNA sequencing analysis the following primers were used: F:5’AGACGCCTGTGTACGACCTT3’ R:5’ AGACTGCTGAGGGGAGACAG3’


0 0.2 0.4 0.6 0.8 1 1.2 1.4

pri-miR-212

pri-miR-613

pri-miR-7-3

pri-miR-425

pri-miR-601

pri-miR-196a

pri-miR-26a1

pri-miR-627

pri-miR-216

pri-let7f

pri-miR-625

pri-miR-375

pri-miR-99a

pri-miR-100

pri-miR-125b1

pri-miR-205

pri-miR-150

pri-miR-125a

pri-miR-10a

pri-miR-125b2

pri-miR-194b1

pri-miR-181a2

pri-miR-30a

pri-miR-7-1

pri-miR-22

pri-miR-31

pri-miR-21

pri-miR-let7a

pri-miR-193b

Fold change

Co115.TRBP wtCo115.pLPC

a c

SKUT1B

H23

Co115

HCT116

TARBP2

Centromere

Chr 12

RKO

TRBP

SKUT1B

H23

Co115

HCT116

TARBP2

Centromere

Chr 12

RKO

TRBP

b

TRBP

β-actin

DICER1RKO-ScramblesiRNA-TRBP

0 0.2 0.4 0.6 0.8 1

Mean

Pre 7

Pre 193b

Pre let7a

Pre 125b

Pre 30a

Pre 22

Pre 31

Pre 21

Pre 212

RKO S

cram

bleRKO

siR

NATR

BP

Supplementary Figure 1. FISH analysis of TRBP, TRBP RNA interference and quantification of primary microRNA trancritps. (a) Metaphases spreadsfrom HCT116, RKO, Co115 and SKUT1B cancer cell lines show two copies of chromosome 12 with red signals, that correspond to the clone spanning theTRBP gene, and green signals, that correspond to the control BAC clone mapping to the 12q13 region. (b) TRBP and DICER1 protein expression levels in RKO cells knocked down by siRNA of TRBP (left panel). TRBP siRNA cells showed a mean of 58% decrease in the processing efficiency of precursor microRNAs(right panel). (c) qPCR of primary microRNA transcripts showed no significant differences between TRBP mutant Co115 cells (Co115.pLPC) and Co115 cellstransfected with the wild-type TRBP (Co115.TRBP wt).Nature Genetics: doi:10.1038/ng.317

a b

Supplementary Figure 2. HeatMap, qPCR validation of microRNA array and downregulation of targets of overexpressed miRs upon TRBP transfection in Co115 cell line. (a) HeatMap of microRNA array showing mostly upregulated microRNAs in Co115 transfected cells compared to empty vector transfectedcells. Absolute expression values (intensities) for each microRNA are represented in the cluster. (b) Upregulated microRNAs were validated by qPCR. Differences in fold change values of Co115 transfected with TRBP wt in comparison to empty vector transfected Co115 are generally more pronounced in qPCR, due to the higher sensitivity of this technique in comparison with the arrays. Downregulated microRNAs were also validated by qPCR. (c) EZH2 and ERBB2 are previously described targets of miR-26a and miR-125a/b respectively. As a consequence of upregulation of these microRNAs, lower protein levels of EZH2 and ERBB2 are shown in TRBP Co115 transfected cells versus empty vector transfected Co115 cells.

0 1 2 3 4 5 6 7 8 9

miR 181a2

miR 194-1

miR 125b2

miR 10a

miR125a

miR 150

miR 205

miR 26a

miR 125b1

miR 100-A

miR 99a

miR 375

miR 100-1/2

miR 625

miR-let7f

miR 216

miR 627

miR 26a1

miR 196a1

miR 601

miR 425-5p

miR 7-3p

miR 613

miR 212

Fold change

Co115.pLPCCo115.TRBP wt

0 0.2 0.4 0.6 0.8 1

miR 106b

miR 16a

miR 181d

miR 321

miR 594

miR 16-1

miR 95

miR 553

miR 801

Fold change

Co115.pLPC

Co115.TRBP wt

c

EZH2

β-actin

Co115

.pLP

CCo1

15.T

RBP wt

ERBB2

Nucleolin

Co115

.pLP

CCo1

15.T

RBP wt

Co1

15.T

RB

P w

tC

o115

.TR

BP

wt

Co1

15.T

RB

P w

t

Co1

15.p

LPC

Co1

15.p

LPC

Co1

15.p

LPC


a b

0

0.5

1

1.5

2

2.5

3

Fold

cha

nge

Co115.Negative control

Co115.Pre125b1

Co115.Pre125b2

Co115.Pre-let7a

miR-125b1 miR-125b2 Let7-a0

0.5

1

1.5

2

2.5

3

Fold

cha

nge


Co115.Pre125b1

Co115.Pre125b2

Co115.Pre-let7a

miR-125b1 miR-125b2 Let7-a 0123456789

10

Doub

ling

time

/ h


Co115.Pre125b1

Co115.Pre125b2

Co115.Pre-let7a

*

**

0123456789

10

Doub

ling

time

/ h


Co115.Pre125b1

Co115.Pre125b2

Co115.Pre-let7a

*

**

Supplementary Figure 3. Transfection of Precursor microRNA molecules 125b1, 125b2 and let-7a. (a) qPCR showing slight increase of the corresponding mature microRNAs in Co115 transfected cell line with pre-miR-125b1, pre-miR-125b2 and pre-let7a. (b) Co115 transfected with “tumor suppressor” precursor molecules showed a significant increased in doubling time. The assay was performed as follows: the same cell number was platted in 12 well plates by triplicate and left to grow for 24, 48, 72 and 96 hours. Cells were tripsinized and counted using a Neubauer chamber at the time points upper mentioned and kept for RNA extraction. The doubling time was calculated as follows: (in hours)=h*ln(2)/ln(c2/c1) (J. M. Davis, Basic Cell Culture: a practical approach).


a d

DICER1_Co115 cell line

-730 -570

DICER1_Co115 cell line

-730 -570

Cycloheximide

DMSO

Co115.TRBP WT

0 2 4 6 8 10

Co115

0 2 4 6 8 10

DICER1

DICER1

Nucleolin

b

RKO RKO

Co11

5

RKO

Co11

5Co

115

Lactacystin 4h MG132 8h 12h

DICER1

Nucleolin

RKO

Centromere

Chr 14/22

DICER1

Co115RKO

eRKO HCT116

Cycloheximide

DMSO

DICER1

DICER1

Nucleolin

0 2 4 6 8 100 2 4 6 8 10

c

Supplementary Figure 4. DICER1 studies. (a) DICER1 promoter CpG island representation. The DICER1 CpG island was found unmethylated in Co115 cells(Bisulfite genomic sequencing primers: Foward:5’TGTTGTAGGTTTAGGTGAATGG 3’ and Reverse:5’ACACAAACCCATAAAAAAATCC 3’). (b) FISH analysis of DICER1. Metaphases spreads from RKO and Co115 cell lines show two copies of chromosome 14 and 22 with red signals that correspond to theclone spanning the DICER1 gene and green signals that correspond to the control BAC clone mapping to the 14q32 region. (c) Proteasome inhibition assay. Cellswere treated with lactacystin and MG132 proteasome inhibitors. DICER1 protein expression in Co115 cells was not restored upon the use of these drugs. (d) Cycloheximide Translation Inhibition. Cells were counted and equally platted in triplicates and treated with 50 µg/ml cycloheximide (dissolved in DMSO). They were harvested at various time points indicated, protein was quantified using Bradford and immunoblotted for DICER1 using nucleolin as a loading control. Co115 and Co115.TRBP wt cells treated in parallel with 50 ug.mL cycloheximide and DMSO as a control. Co115 transfected with wild-type TRBP is able to maintain DICER1 stability after translation inhibition as well as TRBP wt cell lines (RKO and HCT116) (e).Nature Genetics: doi:10.1038/ng.317

a b c

DICER1

TRBP

β-actin

Co115

.pLPC

.pCMV.T

ag4B

Co115

.DIC

ER1+TR

BP

HCT116

DICER1

TRBP

β-actin

Co115

.pLPC

.pCMV.T

ag4B

Co115

.DIC

ER1+TR

BP

HCT116

e f g

Supplementary Figure 5. Tumor supressor-like features of the TRBP/DICER1 complex. (a) TRBP and DICER1 protein expression in Co115 cells transfected with empty vector or double TRBP/DICER1. (b) Co-transfected cells showed a 4.6 fold increase in the processing efficiency of precursor microRNAs in comparison to empty vector transfected cells. (c) TRBP and DICER1 transfected cells showed a very significant increase in the efficiency to process precursor synthetic microRNAs in comparison to empty vector transfected cells. (d) Fold change of overexpressed microRNAs in a microarray in DICER1 and doubleTRBP/DICER1 transfected cells in comparison to empty vector transfected Co115 cells e. (e) The co-expression of TRBP and DICER1 in Co115 cells dramaticallyreduced cell viability in comparison to empty vector transfected cells. (f) Colony formation units (CFU) were significantly reduced in TRBP/DICER1 transfected Co115 cells in comparison to empty vector transfected cells (*p<0,001). (g) Drastic reduction in tumor growth in nude mice xenografted with TRBP/DICER1 cotransfected Co115 cells in comparison to empty vector transfected cells (p=2.99 x10-5).

Empty vector

Co115.DICER1 TRBP wt

0

20

40

60

80

100

120

140

160

*

0 4 8 12

Mean

Pre 30

Pre 31

Pre 7

Pre let7a

Pre 212

Pre 125b

Pre 193b

Pre 21

Pre 22

Processing fold

Co115.pLPC.pCMV.Tag4B

Co115.DICER1+TRBP

*0 4 8 12

Mean

Pre 30

Pre 31

Pre 7

Pre let7a

Pre 212

Pre 125b

Pre 193b

Pre 21

Pre 22

Processing fold


Co115.DICER1+TRBP

* 0 10 20 30 40 50 60

Pre 148a

Pre 30a

Pre 138

Pre 19a

Processing fold


Co115.Dicer1+TRBP

Cel

lvia

bilit

y(r

elat

ive

units

)

Days

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1 2 3 4 5


Co115.DICER1.TRBPw t

Empty vector


0

0,05

0,1

1 2 3 4 5

Weeks

Tum

or v

olum

e (m

m3)


Co115.DICER1+TRBP

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

*


Tum

or w

eigh

t(gr

)

CFU

Empty vector

Empty vector



a b

Supplementary Figure 6. TRBP short harpin experiments, TRBP induced overexpression and DICER1 RNAi. (a) TRBP and DICER1 protein expression levelsin RKO and HCT116 cells transfected with a short harpin RNA for TRBP. We could observe a decrease in DICER1 protein levels in transfected shTRBP cells. (b) TRBP shRNA transfected cells showed a significant decrease in the processing efficiency of precursor microRNAS. (c) The expression of a short harpin targetingTRBP increased cell viability very significantly as also the ability to form colonies. (d) Western blot showing TRBP overexpression clone in RKO cell line. Theoverexpression of TRBP wt in RKO cell line did not show any significant alterations in cell viability measured by the MTT assay (lower panel). (e) DICER1 andTRBP protein expression levels in RKO cells knocked down by siRNA of DICER1. RKO cells knocked down for DICER1 presented a mean 80% decrease in theprocessing efficiency of precursor microRNAs (right panel).

Scramble

shTRBPEmpty

vector

RKO HCT116

Scramble

shTRBPEmpty

vector

TRBP

β-actin

DICER1

RKO shTRBP

RKO empty vector

HCT116 shTRBP

HCT116 empty vector

0 0,2 0,4 0,6 0,8 1

Mean

Pre 212

Pre 22

Pre 125b

Pre let7a

Pre 193b

Pre 7

Pre 21

Pre 30a

Pre 31

Processing fold

c d e

0 0,2 0,4 0,6 0,8 1

Mean

Pre 212

Pre 22

Pre 125b

Pre let7a

Pre 193b

Pre 7

Pre 21

Pre 30a

Pre 31

Processing fold

RKO RKO.TRBP wt

DICER1

Nucleolin

TRBP

RKO-

Scra

mbl

eRK

O-si

RNA

DICE

R1_

1RK

O-s

iRNA

DICE

R1_

11

RKO empty vector RKO shTRBP

HCT116 emptyvector HCT116 shTRBP

RKO-Scramble

RNAi DICER1

0 0,2 0,4 0,6 0,8 1

Mean

Pre 31

Pre 193b

Pre 125b

Pre 7

Pre 21

Pre 30a

Pre 212

Pre let7a

Pre 22

Processing fold

TRBP

β-actin

RKO RKO.TRBP wt

0

0.2

0.4

0.6

0.8

0 1 2 3 4 5

RKO shTRBP

RKO empty vector

Cel

lvia

bilit

y(re

lativ

eun

its)

Days

0

0.2

0.4

0.6

0 1 2 3 4 5

HCT116 empty vectorHCT116 shTRBP

Cel

lvia

bilit

y(re

lativ

eun

its)

Days

TRBP

β-actin

0

0.2

0.4

0.6

0.8

1

0 1 2 3 4 5 6

RKO.pLPC

RKO.TRBP w t

Cel

lvia

bilit

y(re

lativ

eun

its)

Days


N1 N2 N3 N4 N5 T1 T2 T3 T4 T5

Normal colon Tumors TRBPmutated

TRBP

β-actin

N6 N7 N8 N9 N10 T7 T8 T9 T10T6

Normal colon Tumors TRBPmutated

TRBP

β-actin

T2 T3 T4 T5T1 T7 T8 T9 T10T6

Tumors TRBP wt

TRBP

β-actin

Supplementary Figure 7. TRBP protein expression measured by western blot in normal colon mucosa andmicrodissected primary colon tumors according to TRBP mutational status. TRBP mutation is associated withdiminished TRBP protein expression.


supplementary information to the manuscript · rossi, agustin f. fernandez, fatima carneiro, carla...

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