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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’
Nature Genetics: doi:10.1038/ng.317
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
Nature Genetics: doi:10.1038/ng.317
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’
Nature Genetics: doi:10.1038/ng.317
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
Nature Genetics: doi:10.1038/ng.317
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.Negative control
Co115.Pre125b1
Co115.Pre125b2
Co115.Pre-let7a
miR-125b1 miR-125b2 Let7-a 0123456789
10
Doub
ling
time
/ h
Co115.Negative control
Co115.Pre125b1
Co115.Pre125b2
Co115.Pre-let7a
*
**
0123456789
10
Doub
ling
time
/ h
Co115.Negative control
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).
Nature Genetics: doi:10.1038/ng.317
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.pLPC.pCMV.Tag4B
Co115.DICER1+TRBP
* 0 10 20 30 40 50 60
Pre 148a
Pre 30a
Pre 138
Pre 19a
Processing fold
Co115.pLPC.pCMV.Tag4B
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.pLPC.pCMV.Tag4B
Co115.DICER1.TRBPw t
Empty vector
Co115.DICER1 TRBP wt
0
0,05
0,1
1 2 3 4 5
Weeks
Tum
or v
olum
e (m
m3)
Co115.pLPC.pCMV.Tag4B
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
*
Co115.DICER1 TRBP wt
Tum
or w
eigh
t(gr
)
CFU
Empty vector
Empty vector
Co115.DICER1 TRBP wt
Nature Genetics: doi:10.1038/ng.317
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
Nature Genetics: doi:10.1038/ng.317
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.
Nature Genetics: doi:10.1038/ng.317
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