tead and ap1 coordinate transcription and motility · c. homer de novo motif analysis of tead4...
TRANSCRIPT
Cell Reports, Volume 14
Supplemental Information
Tead and AP1 Coordinate Transcription and Motility
Xiangfan Liu, Huapeng Li, Mihir Rajurkar, Qi Li, Jennifer L. Cotton, JianhongOu, Lihua J. Zhu, Hira L. Goel, Arthur M. Mercurio, Joo-Seop Park, Roger J.Davis, and Junhao Mao
Table S5 (Related to Experimental Procedures). List of qPCR primers, shRNA and sgRNA sequences
qPCR primer 5'-3'
ABL2-F GGCAGAGGTATGGTCCTTGG
ABL2-R AAAGCTTCTGGACTACTGCCT
ANKRD1-F CGAGATAAGTTGCTCAGCACAG
ANKRD1-R GTTCAGTCTCACCGCATCATG
AJUBA-F ATATTATGCAGCAGCGGGGA
AJUBA-R CCCCATTGCTTGTAGGATCTTC
AMOTL2 AAAGCAGGTTAAAGGTGCTCCA
AMOTL2 CTCTGTGGGTGCTCTGTCTG
CDH2-F CCGGTTTCATTTGAGGGCAC
CDH2-R CCCATTGAGGGCATTGGGAT
CNN3-F GAAGCGAAGTGCGAGAGACC
CNN3-R TATAAGTTCGCAGAGGATGATGC
CTGF-F TGCCCTCGCGGCTTACCGACTG
CTGF-R TGCAGGAGGCGTTGTCATTGGTAAC
CYR61-F GAGTGGGTCTGTGACGAGGAT
CYR61-R GGTTGTATAGGATGCGAGGCT
DAAM1-F CAAGGTGCAGGAGTGGAGAG
DAAM1-R GTGTCAGCCTCTTCCTGGTC
DOCK1-F CCTAGACGCGGAGTTTCCTG
DOCK1-R ACCTCGGTACCACCCTTCAT
DOCK2-F GATGGGTGACCAGCACTACT
DOCK2-R TCCACAGCTGGAACTCAAAGT
DOCK3-F GGCTGAAAAAGGTCATTACTCCC
DOCK3-R CCGAGGAGTGCCCAAAGTTA
DOCK4-F ATGCTTGAGCAGGCACAGAT
DOCK4-R CTCACAGAAGCAGGTGCTGA
DOCK5-F GAAGCCGTCAAGATCAAGGGG
DOCK5-R GCACTTCTCTGCACTCTGACT
DOCK6-F AAACTGGATGCCCAGGTGAG
DOCK6-R CCGGGAGTCATTCGAGTCCT
DOCK7-F GCTCGGTACGTACACAATAGC
DOCK7-R ACAGAAACGGTCTGAACAACA
DOCK8-F ACCCTAGAAGCCACCGAACC
DOCK8-R ACTGAGGTAGTTGAAGAGAGGGG
DOCK9-F GCATATGCGCGAGCTTTCTT
DOCK9-R ATCATCCCAGCTGCTCATGC
GAPDH-F ATGGGGAAGGTGAAGGTCG
GAPDH-R GGGGTCATTGATGGCAACAATA
GPR126-F GAGCCAAGGTTGGTGCTTTG
GPR126-R GGCCAGTAGTAGCCTTTGGG
ITGB5-F TACCTGGAACAACGGTGGAG
ITGB5-R AAAAGATGCCGTGTCCCCAA
KIBRA-F GAGAAGCGGCAGATGGACC
KIBRA-R TCTGCAGAGAGAGCTGGGAT
LATS2-F CAGGATGCGACCAGGAGATG
LATS2-R GTAGGACGCAAACGAATCGC
MACF1-F GTCTTGTCGGAGTGAGCGAT
MACF1-R ATGTGCTTGCGGACCTTCAT
MKLN1-F CGCTACGGTGCTGACAAGAT
MKLN1-R AGAATCAAGTACTGGGGAGGA
NRP1-F ACGCAAGGCGAAGTCTTTTG
NRP1-R CAGTTGGCCTGGTCGTCAT
PARD3-F AGCAGAAATGCTGCCCTCAG
PARD3-R TCAAAGAGCAGTCGGCTGAA
PHLDB2-F CTCTGCTTGCTCACCAGACA
PHLDB2-R TTTCCCGTTCCCTGGATTCG
PTPN14-F TGCCCTCTTTCACACGGATG
PTPN14-R AGGTGTGCGTTTCCGAGTAG
TEAD1-F CCTCACAAGACGTCAAGCCT
TEAD1-R CTTGGTTGTGCCAATGGAGC
TEAD4-F AGTCAGGCACTGGACAAGC
TEAD4-R GCTGGAGACCTGCTTCCTG
TNS3-F TTAACCTCGAGTCTGCACGC
TNS3-R GGGAATCACCACCTTGGCTT
VGLL4-F GCTGTAATGGGAGGAAACCCA
VGLL4-R AGAGCAGCTTCGCGTTTTT
WTIP-F ATCACACGGTTTTTGCACCA
WTIP-R GCAACGACGACACAGTAGGT
YAP-F CCTCGTTTTGCCATGAACCAG
YAP-R GTTCTTGCTGTTTCAGCCGCAG
Primers of ChIP-qPCR 5’-3’
ANKRD1-F GGTGGTGATCACATCGCTCA
ANKRD1-R GGGGGTGTGATATGTAGGGC
AJUBA-F TCCAGTCCGCAGGTCTATCT
AJUBA-R CTCCACATCCAGGGCAGTTA
AMOTL2 CGGGCTAAGTGAATGCTTCC
AMOTL2 GGGGCGCCTATTATCACCTT
CTGF-F AGGCTTTTATACGCTCCGGG
CTGF-R TGAGTGTCAAGGGGTCAGGA
CYR61-F GCCAACCAGCATTCCTGAGA
CYR61-R GAGCCCGCCTTTTATACGGG
DOCK9-F GAGCAAAATAGCCAAGCGGC
DOCK9-R GCATGGAATCTGGAGTGGACA
KIBRA-F ATGTGGCTGAGCCGGTTTTA
KIBRA-R GGCAAGCAGGGTGAGGTAAT
LATS2-F GTCACTCTCGGCCGCATAC
LATS2-R GGCAGATAGCAGCCAGTCAA
PTPN14-F TTTTAAAAGGTGGGGGCCGA
PTPN14-R TCCTCGGAAGTCTAGGTGGG
TEAD1-F TCCTTTCCCAACCGTAAGACA
TEAD1-R CGCGGAAGTCTGCAAGTGTA
TEAD4-F CTTGGTAGGACTTTGCCGGA
TEAD4-R ATGGGTGCTGGGTCTCTTCT
VGLL4-F AGGAGGACCTCTGTACCTGC
VGLL4-R CCAGCTGCCCAGAGAGTCTA
WTIP-F CTGCGGAAATCCGAGCTCTTC
WTIP-R AGGAATGCGAGGAAGGAGACG
YAP-F GCCGGCTCACGGTATCTATTT
YAP-R AGGCCGAAAGAAGTGGAGAG
shRNA 5’-3’
Tead1/3/4 ATGATCA ACTTCATCCACAAG
YAP AAGCTTTGAGTTCTGACATCC
TAZ AGAGGTACTTCCTCAATCA
sgRNA for Crispr 5’-3’
Dock4 TACAGTTCAGATCCTGGAG
Dock9 GAAAGTCATCGTAAGGGAA
SRC1 GGTCGGATGAACTGTCCCCG
SRC2 GAAAAACGTAATCGTGAAC
SRC3 GAAAAACGGAGACGGGAGC
Tead2 GGAGGGTACCGGCGGCAGTG
Figure S1 (Related to Figure 1, 2)
A B C D E
F G H
Tead
4 IH
CTe
ad4
IHC
0%
50%
100%
Normal peripheralnerve tissue
Neuroblastoma
High expressionMedium expressionLow expression
I
(N=10)(N=50)
Pan-TEAD
TEAD1
TEAD4
GAPDH
J K
CTG
F m
RN
A le
vel (
%)
0
400
800
CaC
o2H
T29
FET
RKO
SW48
DLD
1H
CT1
16LS
174T
JunD:
DN-JunD:
AP1-
Luc
(%)
Anti-Flag
DBD
DBD
L
Flag tag
M
N
0
40
80
120 ControlDN-JunD
A. Homer de novo motif analysis of Tead4 ChIP-seq peaks in A549 cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details
1
1e-818 -1.884e+03 37.85% 5.39% AP1
2
1e-557 -1.284e+03 41.04% 10.17% TEAD
3
1e-195 -4.507e+02 27.55% 10.47% FOXA1
4
1e-71 -1.656e+02 22.97% 12.55% CEBP
5
1e-49 -1.148e+02 11.66% 5.48% NFE2L1::MafG
B. Homer de novo motif analysis of Tead4 ChIP-seq peaks in HCT116 cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details
1
1e-390 -8.995e+02 45.38% 5.10% AP1
2
1e-336 -7.743e+02 41.06% 4.87% TEAD
3
1e-33 -7.700e+01 11.09% 3.42% Tcf4
4
1e-29 -6.795e+01 17.95% 8.09% RUNX2
5
1e-27 -6.387e+01 2.70% 0.19% CTCF
C. Homer de novo motif analysis of Tead4 ChIP-seq peaks in SK-N-SH cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details
1
1e-1520 -3.501e+03 38.20% 5.81% AP1
2
1e-618 -1.425e+03 36.46% 12.53% TEAD
3
1e-250 -5.777e+02 40.17% 22.77% NFE2L1::MafG
4
1e-214 -4.950e+02 14.36% 4.91% RUNX
5
1e-114 -2.632e+02 21.80% 12.42% FEV
D. Homer de novo motif analysis of Tead4 ChIP-seq peaks in ECC1 cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details
1
1e-1799 -4.143e+03 44.98% 12.94% TEAD
2
1e-665 -1.533e+03 13.48% 2.74% AP1
3
1e-210 -4.857e+02 33.84% 22.14% NFIC
4
1e-207 -4.778e+02 31.79% 20.47% SOX9
5
1e-203 -4.691e+02 25.49% 15.31% RELA
E. Homer de novo motif analysis of JunD ChIP-seq peaks in A549 cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match
1
1e-2546 -5.864e+03 40.30% 3.11% AP1
2
1e-181 -4.173e+02 11.97% 4.12% FOXA1
3
1e-114 -2.635e+02 15.56% 7.82% Atf1(bZIP)
4
1e-99 -2.281e+02 26.08% 16.62% TEAD
5
1e-70 -1.628e+02 28.76% 20.31% POL003.1_GC-box
F. Homer de novo motif analysis of JunD ChIP-seq peaks in HCT116 cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match
1
1e-3481 -8.016e+03 56.18% 3.63% AP1
2
1e-116 -2.688e+02 3.26% 0.41% NFE2L1::MafG
3
1e-101 -2.335e+02 16.32% 8.25% ERG(ETS)
4
1e-78 -1.804e+02 11.85% 5.78% Tcf
5
1e-75 -1.740e+02 40.76% 30.13% TEAD
G. Homer de novo motif analysis of JunD ChIP-seq peaks in SK-N-SH cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match
1
1e-4699 -1.082e+04 60.41% 7.18% AP1
2
1e-349 -8.054e+02 25.59% 11.90% TEAD
3
1e-203 -4.679e+02 17.30% 8.38% MafA
4
1e-170 -3.933e+02 9.87% 3.88% RUNX
5
1e-139 -3.222e+02 26.80% 17.31% NF1
H. Homer de novo motif analysis of Fra2 ChIP-seq peaks in A549 cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details
1
1e-6520 -1.502e+04 48.80% 4.01% AP1
2
1e-364 -8.386e+02 14.38% 5.73% FOXA1
3
1e-184 -4.244e+02 23.80% 15.21% TEAD
4
1e-175 -4.036e+02 20.71% 12.85% cEBP-like_subclass
5
1e-78 -1.809e+02 2.71% 0.97% BORIS(Zf)
I. Homer de novo motif analysis of Fra1 ChIP-seq peaks in HCT116 cells
Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details
1
1e-4409 -1.015e+04 61.68% 4.05% AP1
2
1e-130 -3.005e+02 4.86% 1.00% NFE2L1::MafG
3
1e-111 -2.571e+02 25.27% 15.31% ERG(ETS)
4
1e-54 -1.256e+02 13.97% 8.56% TEAD
5
1e-36 -8.374e+01 16.01% 11.18% NF1-halfsite(CTF)
J. Homer de novo motif analysis of Fra2 ChIP-seq peaks in SK-N-SH cells
Rank Motif P-value log P-pvalue % of
Targets % of
Background Best Match/Details
1
1e-5345 -1.231e+04 65.70% 5.21% AP1
2
1e-196 -4.515e+02 25.71% 13.97% TEAD
3
1e-97 -2.255e+02 32.55% 23.00% NF1-halfsite(CTF)
4
1e-90 -2.072e+02 5.81% 2.16% RUNX
5
1e-71 -1.636e+02 52.71% 43.49% Stat3(Stat)
Figure S2 (Related to Figure 1 and 2)
Figure S3 (Related to Figure 4)
inpu
tIP
JunD
JunD
IP:Tead4
Tead4
IgG
Tead4
─ ─ + : JNK-IN-8
CBA
0
50
100
150
Tead
-Luc
(%)
- JNK-IN-8 + JNK-IN-8
YAP/TA
ZDA
PI
ANK-
Luc
(%)
E
Anti-myc
D
0
600
1200
1800
Figure S4 (Related to Figure 5)
mR
NA
leve
l (%
)m
RN
A le
vel (
%)
A
B
0
50
100
150 ControlDN-JunDTeads KD/KODN-JunD + Teads KD/KO
0
40
80
120 ControlDN-JunDTead KD/KOTead KD/KO + DN-JunD
SUPPLEMENTAL FIGURE LEGENDS
Figure S1 (Related to Figure 1 and 2). Tead expression and inhibition of Tead and AP1
activity in cancer cells (A-I) Tead4 protein expression in human normal peripheral nerve tissue
and neuroblastoma tissue microarray samples. (A, B) Representative IHC images of Tead4
staining showing no or low expression of Tead4 proteins in human normal peripheral nerve
tissues (N=10). (C-H) Representative IHC images of Tead4 staining showing medium (C-E) or
high (F-H) expression of Tead4 proteins in human neuroblastoma tissue microarray samples
(N=50). (I) The percentile of low, medium or high level of Tead4 expression in normal
peripheral nerve and neuroblastoma tissues. (J) qPCR analysis of relative mRNA level of CTGF,
a known YAP/Tead target, in various colorectal cancer cell lines, indicating HCT116 as one of
the cell lines with high-level YAP/Tead activity. (K) Immunoblot analysis of overall Tead (Pan-
Tead), Tead1 and Tead4 proteins in HCT116 cells with or without Tead1-4 KD/KO expression.
(L) Diagram of JunD wild type and DN-JunD dominant repressor constructs. DBD: DNA
binding domain. (M) Immunoblot analysis of the expression of full length JunD and DN-JunD
proteins in transfected 293T cells, detected by an anti-flag tag antibody. (L) DN-JunD inhibits
JunD, cJun, Fos and Fra1-induced AP1-Luc activity in transfected 293T cells.
Figure S2 (Related to Figure 1 and 2). De novo motif analysis of Tead4, JunD and Fra1/2
ChIP-Seq peaks in A549, HCT116, SK-N-SH, and ECC1 cells. Enrichment of AP1 motif on
Tead4-occupied cis-regulatory regions in the genome of A549 (A), HCT116 (B), SK-N-SH (C)
and ECC1 (D) cells. Enrichment of Tead motif on JunD-occupied cis-regulatory regions in the
genome of A549 (E), HCT116 (F) and SK-N-SH (G) cells. Enrichment of Tead motif on Fra1/2-
occupied cis-regulatory regions in the genome of A549 (H), HCT116 (I) and SK-N-SH (J) cells.
Figure S3 (Related to Figure 4). JNK inhibition does not affect YAP activity and TEAD-
AP1 interaction in HCT116 cells. (A) Immunoflurescence staining of YAP/TAZ in HCT116
cells with and without JNK-IN-8 treatment. (B)Tead-dependent luciferase reporter activity in
HCT116 cells with and without JNK-IN-8 treatment. (C) JNK inhibition by JNK-IN-8 does not
affect endogenous JunD and Tead4 binding, measured by co-immunoprecipitation. (D)
Immunoblot analysis of the expression of the wild type cJun and mutated cJun4A proteins in
transfected 293T cells, detected by an anti-myc tag antibody. (E) Relative luciferase reporter
activity of ANK-Luc in HCT116 cells with cJun, cJun4A, or together with Fra1.
Figure S4 (Related to Figure 5). Transcription of the DOCK family factors and selected
Tead target genes in HCT116 cells with or without AP1 and Tead inhibition. (A) qPCR
analysis of transcription of the DOCK family guanine nucleotide exchange factors in HCT116
cells expressing Teads KD/KO, DN-JunD, or both. (B) qPCR analysis of transcription of the
selected Tead targets, including ABL2, CDH2, CNN3, DAAM1, GRP126, ITGB5, MACF1,
MKLN1, NRP1, PARD3, PHLDB2 and TNS3, in HCT116 cells expressing Teads KD/KO, DN-
JunD, or both.
Table S1 (Related to Figure 1). Tead4 ChIP-seq analysis in A549, HCT116, SK-N-SH, and
ECC1 genomes
Table S2 (Related to Figure 2). JunD ChIP-seq analysis in A549, HCT116, and SK-N-SH
genomes
Table S3 (Related to Figure 2). Fra1/2 ChIP-seq analysis in the A549, HCT116, and SK-N-
SH genomes
Table S4 (Related to Figure 5). List of the genes with Tead and AP1 co-occupied sites
identified in A549, HCT116 and SK-N-SH cancer cells.
Table S5 (Related to Experimental Procedures). List of qPCR primers, shRNA and sgRNA
sequences.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Cell culture, transfection, treatment and lentiviral infection. HCT116, SK-N-SH, ECC1, FET,
DLD1, LS174T, HEK293T cells were cultured in DMEM supplemented with 10% FBS. A549
cells were cultured in F-12K Medium supplemented with 10% FBS. HT29 cells were cultured in
McCoy 5A medium supplemented with 10% FBS. Caco2 and RKO cells were cultured in
EMEM medium supplemented with 20% FBS. SW48 cells were cultured in L15 medium
supplemented with 10% FBS. Transfection was performed using Lipofectamine 2000
(Invitrogen). Cells were treated with 1uM JNK Inhibitor JNK-IN-8 (EMD Millipore) or 20nM
Bufalin (Sigma) for 24 hours before subsequent analysis. For lentiviral infection, pLX or pGIPZ
based constructs were transfected along with the packing plasmids into growing HEK293T cells.
Viral supernatants were collected 48 hours after transfection, and target cells were infected in the
presence of polybrene and underwent selection with puromycin for 3-4 days. The lentiviral
expression vectors for Tead1, Tead2, SRC3, Fos, JunD, and Fra1 were purchased from DNASU
Plasmid Repository (https://dnasu.org). cJunWT-Myc and cJun4A-Myc were a gift from Axel
Behrens (Addgene plasmids #47443 and 47444). The cDNA fragments encoding human JunD,
DN-JunD, SRC3, SRC3-N, SRC3-M and SRC3-C were PCR cloned into a pLX or pGIPZ-based
lentiviral vector, and sgRNAs targeting human JunD, cJun, Tead2, SRC1/2/3, and Dock4/9 were
cloned into the pLentiCRISPR-V2 (a gift from Feng Zhang, Addgene plasmid # 52961). For
generation of pLKO-shTead1/3/4, shRNA against human Tead1/3/4 was cloned into pLKO.1-
blast (a gift from Keith Mostov, Addgene plasmid # 26655). The shRNA and sgRNA used for
targeting Teads, YAP/TAZ, SRC and Dock proteins were described in Supplemental Table S5.
Tead4, JunD, Fra1, Fra2, H3K4me1, H3K4me3, and H3K27ac ChIP-seq, de novo motif
discovery and functional clustering analyses. Tead4, JunD, Fra1, Fra2, H3K4me1, H3K4me3
and H3K27ac ChIP-seq data of A549, HCT116, SK-N-SH and ECC1 cells were downloaded
from the ENCODE project (http://genome.ucsc.edu/ENCODE/downloads.html). The antibodies
used in the ChIPseq analysis are as follows: Tead4 (SCBT, sc-101184), JunD (SCBT, sc74),
Fra1 (SCBT, sc183), Fra2 (SCBT, sc604), H3K4me1 (Abcam, ab8895), H3K4me3 (Abcam,
ab8580) and H3K27ac (Abcam, ab4729) Peak calling and de novo motif analyses were done
using Homer (http://homer.salk.edu/homer/). To evaluate the statistical significance of Tead4,
JunD, Fra1/2 ChIP-seq peak overlaps, we sampled random matched genomic regions with the
same number of peaks and same peak length distribution as the identified peaks, and repeated the
process 100 times to obtain the empirical null distribution of the number of overlaps between
identified sites and random regions. The Tead4, JunD, H3K4me3 and H3K27ac tracks of the
ANKRD1, Dock9 and Tead4 loci were generated by Bioconductor package trackViewer
developed by J Ou and LJ Zhu. Functional annotation and clustering analyses of the list of the
genes carrying Tead4 and/or AP1 peaks were done using Database for Annotation, Visualization
and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/).
Immunohistochemistry, immunofluorescence and immunoblotting. Immunohistochemistry
was performed on 5μm sections of formalin fixed and paraffin embedded tumors. Following
rehydration, antigen retrieval was performed at 100 °C for 0.5-2 hours with Tris-EDTA buffer.
Endogenous peroxidase activity was inhibited by incubating with 3% peroxide solution for 20
minutes. Sections were then blocked in a blocking buffer containing 5% BSA and 0.1% Triton
X-100, and incubated overnight at 4℃ in primary antibodies against Tead4 (Abcam, ab97460,
1:100) and JunD (Cell signaling, 5000S, 1:50). Signal detection was accomplished with
biotinylated secondary antibodies in the Vectastain ABC kit (Vector Labs).
For immunofluorescence, cells were fixed by 4% paraformaldehyde for 5 minutes, washed with
PBS and blocking buffer (PBS with 5% BSA and 0.1% Triton X-100), and then incubated
overnight at 4℃ in primary antibodies against JunD (Abcam, ab134067), Fra1 (SCBT, SC183),
YAP/TAZ (Cell signaling, 8418s), and SRC1 (Cell signaling, 2191). Alexa-Fluor fluorescent
conjugated secondary antibodies (Invitrogen) were used for detection.
Immunoblotting was performed using standard protocol. The primary antibodies used were: Flag
(Sigma, F3165), Myc (Developmental Studies Hybridoma Bank), V5(Thermo Fisher, MA5-
15253),YAP (Cell signaling, 4912), p-YAP (Cell signaling, 4911), TAZ (BD, 560235)), Tead4
(Abcam, ab97460), pan-Tead (Cell signaling, 13295s), JunD (Abcam, ab134067), cJun (Cell
signaling, 9165), p-cJun (Cell signaling, 9164), Fra1 (SCBT, sc183), Fra2 (SCBT, sc604),
ANKRD1 (Abnova, H00027063-B02P), Tead1 (Abcam, ab133533), Tead2 (Cell Signaling, 887),
Dock4 (Bethyl, A302-263A-T), Dock9 (Bethyl, A300-530A-T), RhoA (Cytoskeleton),
RAC1(Cytoskeleton), SRC1 (Cell signaling, 2191), SRC2 (Bethyl, A300-346A-T), SRC3 (Cell
signaling, 2126) and GAPDH (Santa Cruz, 5147). HRP conjugated secondary antibodies used for
detection were obtained from Jackson Laboratories.
Soft agar colony formation, cell migration and invasion, and small G protein activation. For anchorage-independent soft-agar colony formation assay, cells were seeded at a density of
6000 cells/well in a 6-well plate of 0.3% agarose in DMEM media containing 10% FBS.
Colonies from 12 fields of view were counted 14 days later. Assays were conducted in triplicates,
and standard deviation was used to calculate error bars. For cell scratch assay, the cell monolayer
was scratched in a straight line with a p200 pipet tip, and images were taken at 0 or 48 hours
later. For transwell migration assays, 5X104 cells were plated in the top chamber with the non-
coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). For Matrigel invasion
assays, 2X105 cells were plated in the top chamber with Matrigel-coated membrane (24-well
insert; pore size, 8 mm; BD Biosciences). In both assays, cells were plated in medium without
serum or growth factors, and medium supplemented with serum was used as a chemoattractant in
the lower chamber. The cells were incubated for 24h and cells that did not migrate or invade
through the pores were removed by a cotton swab. Cells on the lower surface of the membrane
were stained with the Diff-Quick Staining Set (Dade) and counted. RhoA/RAC1/CDC42 G-
LISA activation assays (Cytoskeleton) were performed according to the manufacturer’s protocol.
Immunoprecipitation and chromatin immunoprecipitation. HCT116 cells or transfected
HEK293T cells were lysed using lysis buffer (50 mM Tris HCl, pH 8.0, 170 mM NaCl, 0.5%
Nonidet P-40, 50 mM NaF and 1 mM PMSF). Cell lysates were centrifuged at 14,000 × g for 10
minutes, and supernatants were used for immunoprecipitation. After preclearing for 1 hour with
protein A/G-Sepharose (Invitrogen), supernatants were incubated at 4℃ overnight with
antibodies and protein A/G-Sepharose beads. Immunoprecipitates were washed five times with
lysis buffer, and proteins were eluted with SDS sampling buffer, and analyzed by
immunoblotting using the antibodies as indicated. Trueblot secondary antibodies (Rockland)
were used for immunoblot detection.
Chromatin immunoprecipitation ChIP assays were performed according to the manufacturer’s
instructions (Active Motif, CA). Briefly, 2×107 cells were fixed with 1% formaldehyde, washed
with cold PBS and lysed in lysis buffer. After sonication, protein-DNA complexes were
incubated with antibody-coupled protein G beads at 4℃ overnight. After elution in 1%
SDS/0.1M NaHCO3 and reverse cross-link at 65℃, DNA was purified by phenol/chloroform
extraction and ethanol precipitation, and subjected to PCR or qPCR analysis. The primers used
for real-time PCR of cis-regulatory elements were listed in Table S5. In the Re-ChIP
experiments, complexes were eluted by incubation for 30 minutes at 37℃ in 10mM DTT. After
centrifugation, the supernatant was diluted 20 times with Re-ChIP buffer (1% Triton X-100,
2mM EDTA, 150mM NaCl, 20Mm Tris-HCl, pH 8.1) for further ChIP-qPCR analysis. The
primers used for ChIP-qPCR analysis were described in Supplemental Table S5.
Quantitative Real-Time PCR. RNA of cultured cells was isolated using Trizol reagent
(Invitrogen), followed by reverse-transcription using SuperscriptII Reverse Transcriptase
(Invitrogen). Quantitative real-time PCR was performed using Sybr Mastermix (Kapa
Bioscience). The primers used for real-time PCR were described in Supplemental Table S5. All
qPCR experiments were conducted in biological triplicates, error bars represent mean ±
standard deviation, and Student’s t-test was used to generate p-values.
Luciferase reporter analysis. Tead-Luc (8xGTIIC-luciferase) was a gift from Stefano Piccolo
(Addgene plasmid # 34615). AP1-Luc (3xAP-1 in pGL3-basic) was a gift from Alexander Dent
(Addgene plasmid # 40342). The ANK-Luc, Dock9-Luc, Tead4-Luc, and ANK-Luc mutated
constructs were generated by PCR and subcloning into pGL3 based luciferase vectors (Promega).
Cells were transfected with different reporter constructs together with a Renila luciferase
expression plasmid for 48 hours. Luciferase activity was measured as using the Dual-Luciferase
reporter assay system (Promega) according to the manufacturer’s protocol. All luciferase
reporter experiments were conducted in biological triplicates, error bars represent mean ±
standard deviation, and Student’s t-test was used to generate p-values.