SUPPLEMENTAL MATERIAL

A YAP/TAZ-induced feedback mechanism regulates

Hippo pathway homeostasis

Toshiro Moroishi,1 Hyun Woo Park,1 Baodong Qin,1,2 Qian Chen,3 Zhipeng Meng,1 Steven

W. Plouffe,1 Koji Taniguchi,4 Fa-Xing Yu,5 Michael Karin,4 Duojia Pan,3 and Kun-Liang

Guan1,*

1Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, CA

92093, USA

2Department of Laboratory Medicine, Shanghai Changzheng Hospital, Second Military Medical University,

Shanghai 200003, China.

3Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore,

Maryland 21205, USA

4Departments of Pharmacology and Pathology, University of California, San Diego, La Jolla, California

92093, USA

5Children's Hospital and Institute of Biomedical Sciences, Fudan University, Shanghai 200032, China

*Correspondence: kuguan@ucsd.edu

Figures S1 to S7

Supplemental figure legends

Supplemental experimental procedures

SUPPLEMENTAL FIGURE LEGENDS

Figure S1. Rescue experiments and knockdown efficiency for shRNA studies, effects of

YAP/TAZ on each other's mRNA levels, related to Figure 1

(A) YAP re-expression abolishes TAZ accumulation in YAP knockdown MCF10A cells.

MCF10A cells stably expressing shRNAs specific for YAP were infected with retroviruses

encoding Myc-YAP. Immunoblot analysis (IB) was performed with the indicated

antibodies.

(B) Knockdown efficiency of TEAD1/3/4 shRNA. Total RNA extracted from MCF10A

cells infected with lentiviral vectors encoding shRNAs specific for TEAD1/3/4 (or control)

was subjected to RT and real-time PCR analysis of the indicated mRNA. Data are means ±

SD of triplicates from a representative experiment.

(C) TEAD1 re-expression stimulates YAP-induced TAZ reduction in TEAD1/3/4

knockdown MCF10A cells. MCF10A cells stably expressing Myc-YAP(5SA) and shRNAs

specific for TEAD1/3/4 were infected with retroviruses encoding Myc-TEAD1.

Immunoblot analysis was performed with the indicated antibodies.

(D) Overexpression of YAP has little effect on the mRNA abundance of TAZ. Total RNA

extracted from MCF10A cells stably expressing Myc-YAP(5SA), Myc-YAP(5SA/S94A), or

control vector was subjected to RT and real-time PCR analysis of WWTR1 mRNA. Data are

means ± SD of triplicates from a representative experiment.

(E) Overexpression of TAZ has little effect on the mRNA abundance of YAP. Total RNA

extracted from MCF10A cells stably expressing FLAG-TAZ(4SA),

FLAG-TAZ(4SA/S51A), or control vector was subjected to RT and real-time PCR analysis

of YAP1 mRNA. Data are means ± SD of triplicates from a representative experiment.

Figure S2. Effect of YAP/TAZ on mRNA level of MYC or AMOT, related to Figure 2

(A) Overexpression of YAP/TAZ does not increase the mRNA abundance of MYC. Total

RNA extracted from MCF10A cells stably expressing Myc-YAP(5SA),

Myc-YAP(5SA/S94A), FLAG-TAZ(4SA), FLAG-TAZ(4SA/S51A), or control vector was

subjected to RT and real-time PCR analysis of MYC mRNA. Data are means ± SD of

triplicates from a representative experiment.

(B) Overexpression of TAZ has little effect on the mRNA abundance of AMOT. Total RNA

extracted from MCF10A cells stably expressing FLAG-TAZ(4SA),

FLAG-TAZ(4SA/S51A), or control vector was subjected to RT and real-time PCR analysis

of AMOT mRNA. Data are means ± SD of triplicates from a representative experiment.

Figure S3. TAZ induces LATS2 expression, knockdown efficiency of YAP shRNA,

related to Figure 3

(A) TAZ induces the transcription of LATS2, but not that of LATS1. Total RNA extracted

from MCF10A cells stably expressing FLAG-TAZ(4SA), FLAG-TAZ(4SA/S51A), or

control vector was subjected to RT and real-time PCR analysis of the indicated mRNA.

Data are means ± SD of triplicates from a representative experiment.

(B) Knockdown efficiency of YAP shRNA. Total RNA extracted from MCF10A cells

infected with lentiviral vectors encoding shRNAs specific for YAP (or control) was

subjected to RT and real-time PCR analysis of YAP1 mRNA. Data are means ± SD of

triplicates from a representative experiment.

Figure S4. YAP and TAZ stimulate the intrinsic kinase activity of LATS1/2 through

NF2 induction, related to Figure 4

(A) MST1/2 re-expression prevents YAP dephosphorylation in MST1/2 deficient

HEK293A cells. Wild-type (WT) and MST1/2 double knockout (DKO) HEK293A cells

transfected (or not) with expression vectors for the indicated protein were subjected to

immunoblot (IB) analysis. Where indicated, gels containing phos-tag were employed for

assessment of YAP phosphorylation status (top).

(B) TAZ activates LATS. HEK293A cells stably expressing FLAG-TAZ(4SA), FLAG-

TAZ(4SA/S51A), or control vector were subjected to immunoblot analysis. HM,

hydrophobic motif.

(C) MST1/2 are largely required for YAP-induced activating phosphorylation of LATS1/2.

WT and MST1/2 DKO HEK293A cells infected with retroviruses encoding Myc-

YAP(5SA) (or control empty retrovirus) were subjected to immunoblot analysis.

(D) YAP and TAZ induce the transcription of NF2. Total RNA extracted from MCF10A

cells stably expressing the indicated constructs were subjected to RT and real-time PCR

analysis of NF2 mRNA. Data are means ± SD of triplicates from a representative

experiment.

(E) Both endogenous YAP and TEAD1 bind to the NF2 promoter. MCF10A cells starved in

serum-free medium for 18 h were stimulated with 5% horse serum for 2 h. The cells were

then subjected to chromatin immunoprecipitation (ChIP) with antibodies to endogenous

YAP or TEAD1 (or control IgG), and the precipitated DNA was quantitated by real-time

PCR analysis with primers specific for a promoter region or a control region (CR) of the

indicated genes. Data are means ± SD of triplicates from a representative experiment.

(F) NF2 re-expression prevents YAP dephosphorylation in NF2 deficient HEK293A cells.

Wild-type (WT) and NF2 knockout (KO) HEK293A cells transfected (or not) with

expression vectors for the indicated protein were subjected to immunoblot analysis. Where

indicated, gels containing phos-tag were employed for assessment of YAP phosphorylation

status (top).

(G) NF2 is required for YAP-induced LATS activation. WT and NF2 KO HEK293A cells

infected with retroviruses encoding Myc-YAP(5SA) (or control empty retrovirus) were

subjected to immunoblot analysis.

(H) NF2 is required for TAZ-induced activating phosphorylations of LATS1/2, but not for

induction of LATS2. WT and NF2 KO HEK293A cells were infected with retroviruses

encoding FLAG-TAZ(4SA), or with the empty retrovirus. Cell lysates were immunoblotted

with the indicated antibodies.

(I) LATS2 induction by YAP(5SA) is still operational under NF2 and MST1/2

overexpression. Tetracycline repressor-expressing HEK293A cells were infected with

retroviruses encoding Myc-YAP(5SA) (or with the control empty retrovirus), transfected

(or not) with expression vectors for the indicated protein, and then incubated with

doxycycline (200 ng/ml) for 28 h. Immunoblot analysis was performed with the indicated

antibodies.

Figure S5. LATS1/2 mediate negative feedback regulation of YAP, TAZ, and AMOT,

related to Figure 5

(A) LATS1/2 are required for YAP-induced TAZ reduction in MCF10A cells. MCF10A

cells stably expressing Myc-YAP(5SA) (or control vector) were transfected with non-

targeting siRNA (control), or siRNA targeting LATS1 or LATS2. Cell lysates were

collected for immunoblot (IB) analysis.

(B) YAP induces the transcription of Lats2. Wild-type (WT) or Lats1–/–Lats2Δ/Δ (LATS1/2

DKO) MEFs infected with retroviruses encoding Myc-YAP(5SA) (or with the empty

retrovirus) were subjected to RT and real-time PCR analysis of Lats2 mRNA. Data are

means ± SD of triplicates from a representative experiment.

(C) LATS1/2 are required for YAP-induced phosphorylation and accumulation of AMOT.

MEFs infected as in (A) were subjected to immunoblot analysis with antibodies to the

indicated proteins.

Figure S6. TEADs are required for lysophosphatidic acid (LPA)-induced CTGF and

CYR61 transcription, related to Figure 6

(A) LPA induces YAP/TAZ target gene expression in a TEAD-dependent manner. MCF10A

cells stably expressing shRNA specific for TEAD1/3/4 (or control) were starved in serum-

free medium for 15 h and then stimulated with 5 μM LPA for 2 h. Total RNA were

subjected to RT and real-time PCR analysis of the indicated mRNA. Data are means ± SD

of triplicates from a representative experiment.

Figure S7. Rescue experiment for LATS1/2 double knockout HEK293A cells, related

to Figure 7

(A) LATS1/2 re-expression prevents YAP dephosphorylation in LATS1/2 deficient

HEK293A cells. Wild-type (WT) and LATS1/2 double knockout (DKO) HEK293A cells

transfected (or not) with expression vectors for the indicated protein were subjected to

immunoblot (IB) analysis. Where indicated, gels containing phos-tag were employed for

assessment of YAP phosphorylation status (top).

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Cell culture and transfection

All cell lines were cultured under an atmosphere of 5% CO2 at 37°C. MCF10A cells were

cultured in DMEM/F12 (Invitrogen) supplemented with 5% horse serum (Invitrogen),

epidermal growth factor (EGF; 20 ng/ml), hydrocortisone (500 ng/ml), insulin (10 μg/ml),

cholera toxin (100 ng/ml), penicillin (100 U/ml), and streptomycin (100 mg/ml). All other

cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Thermo

scientific), penicillin (100 U/ml), and streptomycin (100 mg/ml). For serum starvation, cells

were incubated in DMEM or DMEM/F12 without other supplements. Transfection of

plasmid DNA was performed with the PolyJet DNA In Vitro Tranfection Reagent

(Signagen Laboratories) according to manufacturer’s instructions. Cloning and construction

of the expression plasmids were described previously (Zhao et al. 2007; Zhao et al. 2008).

The empty pcDNA3 plasmid was included to ensure that cells were transfected with equal

amounts of total DNA. Small interfering RNA (siRNA) was delivered into cells using

Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instructions.

Retroviral infection and tetracycline-inducible expression system

Cells stably expressing empty vector, Myc-YAP, Myc-YAP(5SA), Myc-YAP(5SA/S94A),

FLAG-TAZ(4SA), FLAG-TAZ(4SA/S51A), or Myc-TEAD1 were generated by retroviral

infection. 293 phoenix retrovirus packaging cells were transfected with empty vector,

pQCXIH Myc-YAP, pQCXIH Myc-YAP(5SA), pQCXIH Myc-YAP(5SA/S94A), pBABE

FLAG-TAZ(4SA), pBABE FLAG-TAZ(4SA/S51A), or pQCXIH Myc-TEAD1 constructs.

Forty-eight hours after transfection, retroviral supernatant was supplemented with 5 μg/ml

polybrene, filtered through a 0.45 μm filter, and used to infect the indicated cells. Forty-

eight hours after infection, cells were selected with either 200 μg/ml hygromycin (for

pQCXIH constructs) or 4 μg/ml puromycin (for pBABE constructs) in culture medium.

For tetracycline-inducible expression system, MCF10A and HEK293A cells were

infected with a retrovirus encoding tetracycline repressor (TetR) (pRetroX-Tet-On;

Invitrogen) for 2 days. The cells were then incubated with G418 (300 μg/ml) for selection

and amplification of neomycin-resistant cells. The TetR-expressing cells were then infected

with retroviruses encoding YAP(S127A) or Myc-YAP(5SA) (or pRetroX-Tight-Pur empty

vector for control). Forty-eight hours after infection, the cells were selected with G418 (300

μg/ml) and puromycin (4 μg/ml) in culture medium. For induction of YAP expression, the

cells were incubated with doxycycline for the indicated times.

Lentiviral Infection and RNA Interference (RNAi)

Gene silencing by RNAi was performed with either the lentivirus-based shRNA expression

or siRNA transfection. For lentiviral infection, the infection process was similar to that of

retroviral infection except that the lentiviral packaging plasmids psPAX2 and pMD2.G

were cotransfected into HEK293T cells for virus production. The shRNA-encoding DNA

oligonucleotide inserts were generated with the use of pLKO.1 vector. The target sequences

for YAP and TEAD1/3/4 were described previously (Zhao et al. 2008). An shRNA specific

for LacZ (5'-AAGGCCAGACGCGAATTAT-3'), which did not match any existing

sequence in the human database, was used as a control. For siRNA experiments, ON-

TARGETplus SMARTpool siRNA (GE Dharmacon) for LATS1 (L-004632-00), LATS2 (L-

003865-00), or non-targeting control (D-001810-10) were used.

Gene deletion of HEK293A cells by CRISPR/Cas9 system

LATS1/2-, MST1/2-, or NF2-deficient HEK293A cells were created through the CRISPR

(clustered regularly interspaced short palindromic repeats)/Cas9 system (Ran et al. 2013).

HEK293A cells were transfected with a Cas9 expression plasmid together with single-guide

RNA (sgRNA) expression plasmids (PX459; Addgene plasmid #48139). The guide

sequences were designed using the CRISPR design tool at http://www.genome-

engineering.org/crispr or were taken from archived guide sequences from the Genome-

scale CRISPR knock-out (GeCKO2) library (Sanjana et al. 2014). The guide sequences

used are 5'-CGTGCAGCTCTCCGCTCTAA-3' for human LATS1; 5'-

TACGCTGGCACCGTAGCCCT-3' for human LATS2; 5'-

ATACACCGAGATATCAAGGC-3' for human MST1; 5'-AGTACTCCATAACAATCCAG-

3' for human MST2; 5'-GTCCATGGTGACGATCCTCA-3' for human NF2. Following

transfection and selection with puromycin, cells were single-cell sorted by fluorescence-

activated cell sorting (FACS) into 96-well plate format. Knockout clones were selected by

immunoblot analysis, and genomic mutations were confirmed by genome sequencing. Two

independent clones were analyzed as indicated.

Gene deletion of MEFs by adenoviral infection

SV40 largeT-immortalized Lats1–/–Lats2F/F MEFs were described previously (Kim et al.

2013). For Lats2 deletion, MEFs were infected with an adenovirus encoding Cre

recombinase (#1700, Vector Biolabs).

Immunoprecipitation and immunoblot analysis

Cells were lysed with a buffer comprising 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5%

Triton X-100, 1 mM PMSF, protease inhibitor cocktail (Roche), 400 μM Na3VO4, 400 μM

EDTA, 10 mM NaF, and 10 mM sodium pyrophosphate. The lysates were centrifuged at

16,000 × g for 10 min at 4°C to remove debris, and the resulting supernatants were adjusted

with lysis buffer to achieve a protein concentration of 1.5 mg/ml. The supernatants were

then incubated for 2 h at 4°C with primary antibodies and protein A–Sepharose 4 Fast Flow

(GE Healthcare). The immunoprecipitates were washed three times with lysis buffer and

were then subjected to immunoblot analysis with primary antibodies and HRP-conjugated

secondary antibodies to either mouse, rabbit, or goat IgG. The phos-tag electrophoresis was

performed as described previously (Yu et al. 2012). YAP proteins can be separated into

multiple bands in the presence of phos-tag depending on differential phosphorylation

levels, with phosphorylated proteins migrating more slowly.

Antibodies

Antibodies to YAP (#4912), pYAP (S127 in humans and S112 in mice; #4911), TAZ

(#4883), YAP/TAZ (#8418), LATS1 (#3477), LATS2 (#5888), pLATS1/2 (HM; #8654),

pLATS1/2 (AL; #9157), MST1 (#3682), NF2 (#12888), pMOB1 (T35; #8699), pERK1/2

(T202/T204; #4370), and ERK1/2 (#4695) were obtained from Cell Signaling; those to

YAP (ab52771), MST2 (ab52641), and actin (ab3280) were from Abcam; those to CTGF

(sc-14939) and CYR61 (sc-13100) were from Santa Cruz; those to TAZ (#560235) and

HSP90 (#610418) were from BD biosciences; those to LATS1 (A300-478A), LATS2

(A300-479A) and AMOT (A303-305A) were from Bethyl Laboratories. GST (13-6700)

antibody was from Invitrogen. pAMOT (S175 in humans and S176 in mice) antibody was a

gift from Dr. Bin Zhao.

Immunostaining

MCF10A cells and MEFs cultured on glass coverslips, were prepared for immunostaining.

Cells were fixed for 10 min at room temperature with 4% paraformaldehyde in phosphate-

buffered saline (PBS) and were permeabilized with 0.1% Triton X-100 in PBS for 10 min at

room temperature. Cells were then incubated consecutively with primary antibodies

(overnight at 4°C) and Alexa Fluor 488-labeled goat secondary antibodies (for 90 min at

room temperature) in PBS containing 1% bovine serum albumin (BSA). For staining of F-

actin, cells were incubated with 6.6 nM Alexa Fluor 488-phalloidin for 90 min after

blocking. Cells were covered with a drop of ProLong Gold antifade reagent with DAPI

(Invitrogen) for observation.

Kinase assay

The immunoprecipitated LATS1 were washed two times with lysis buffer, followed by once

with wash buffer (40 mM HEPES pH 7.5, 200 mM NaCl) and once with kinase assay

buffer (30 mM HEPES pH 7.5, 50 mM potassium acetate, 5 mM MgCl2). The

immunoprecipitates were then subjected to a kinase assay in 30 μl of kinase assay buffer

supplemented with 500 μM ATP, and 1 μg of GST-YAP expressed and purified from

Escherichia coli as substrates. The reaction mixtures were incubated for 30 min at 30°C,

terminated with SDS sample buffer, and subjected to immunoblot analysis. The same

procedure was used for MST1 and MST2 kinase assay, except that GST-MOB1A was used

as substrates.

RNA extraction, reverse transcription (RT), and real-time PCR analysis

Total RNA (1 μg) isolated from cells with the use of RNeasy Plus Mini Kit (Qiagen) was

reverse-transcribed to complementary DNA using iScript reverse transcriptase (Bio-Rad).

Complementary DNA was then diluted and used for quantification by real-time PCR, which

was performed using KAPA SYBR FAST qPCR master mix (Kapa Biosystems) and the

7300 real-time PCR system (Applied Biosystems). The sequences of the PCR primers

(forward and reverse, respectively) are 5'-GCAAATTCCATGGCACCGT-3' and 5'-

TCGCCCCACTTGATTTTGG-3' for human GAPDH; 5'-

GCCTGGAGAAACCTGCCAAGTATG-3' and 5'-

GAGTGGGAGTTGCTGTTGAAGTCG-3' for mouse Gapdh; 5'-

CCAAGGCTTGACCCTCGTTTTG-3' and 5'-TCGCATCTGTTGCTGCTGGTTG-3' for

YAP; 5'-TCACCAACACCAGCAGCAGATG-3' and 5'-

GGATTCTCTGAAGCCGCAGTTTC-3' for TAZ; 5'-CCCATTCCAGGGTTTGAGC-3'

and 5'-TGCACGAAGAGGTGTTTGTTG-3' for TEAD1; 5'-

GCCCGCTACATCAAGCTGA-3' and 5'-TGGTTGCCATTGTCTGGAAAG-3' for

TEAD2; 5'-GCCAGTGTCCTGCAGAACAA-3' and 5'-

CAAAGGGCTTGATGTCCTGAG-3' for TEAD3; 5'-TCTCTGCCTTCCTGGAGCA-3'

and 5'-TCATAGATTTGGCGGATGTCC-3' for TEAD4; 5'-

TGCCATTGTTTCCAGAGCCCAG-3' and 5'-GCCACCTTCTCATAGCATCCTTCC-3' for

AMOT; 5'-CCTGAGGGAACCGCTTCAAATG-3' and 5'-

TGACTCGTATGGAGGAACAGATGGG-3' for human LATS1; 5'-

CTTCTCTTCACATCCCTCCTCAAGC-3' and 5'-

AGCCTTTATCTCATCAGCACCGTTC-3' for mouse Lats1; 5'-

TCATCCACCGAGACATCAAGCC-3' and 5'-TTGTGAGTCCACCTGAACCCAGTG-3'

for human LATS2; 5'-ATCCTCCCAAAGGGTACAGCACAG-3' and 5'-

TGGTGGCGTCTTGTTCTGGAAG-3' for mouse Lats2; 5'-

CCAATGACAACGCCTCCTG-3' and 5'-TGGTGCAGCCAGAAAGCTC-3' for CTGF; 5'-

AGCCTCGCATCCTATACAACC-3' and 5'-TTCTTTCACAAGGCGGCACTC-3' for

CYR61; 5'-CATACATCCTGTCCGTCCAAGCAG-3' and 5'-

TTTCAACTGTTCTCGTCGTTTCCG-3' for MYC; 5'-

AGCTTCAACCTCATTGGTGACAGC-3' and 5'-TGAGTTCATTGAGCTGCTCCTGC-3'

for NF2. Reactions for GAPDH mRNA were performed concurrently on the same plate as

those for the test mRNAs, and results were normalized by the corresponding amount of

GAPDH mRNA.

Luciferase assay

HEK293A cells were seeded in 24-well plates, and pGL3 LATS2-luciferase reporter,

pCMV FLAG-YAP, pRK7 Myc-TEAD1, and Renilla plasmids were cotransfected as

described above. Luciferase assay was performed 30 h after transfection using the Dual Glo

Luciferase System (Promega). All luciferase activities were normalized to Renilla. A 1000-

bp fragment of human genomic DNA containing promoter lesion of LATS2 was cloned into

the pGL3-Basic vector. Complementary DNA encoding human TEAD1 tagged at its N

terminus with Myc was subcloned into pRK7 vectors. The pCMV-Flag-YAP was kindly

provided by Dr. Marius Sudol.

Chromatin immunoprecipitation (ChIP) assay

MCF10A cells were dual crosslinked consecutively with 2 mM disuccinimidyl glutarate

(DSG; for 45 min) and 1% formaldehyde (for 10 min), and quenched with 0.125 M glycine

for 5 min at room temperature. Harvested cells were washed twice with ice-cold PBS and

then lysed in ChIP cell lysis buffer (20 mM Tris-HCl pH 8.0, 85 mM KCl, 0.5% NP-40).

The lysates were centrifuged at 2,300 × g for 5 min at 4°C to pellet the nuclei, and the

chromatin DNAs were then digested with micrococcal nuclease (MNase) in ChIP MNase

buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 5 mM CaCl2, 0.1% NP-40,

1 mM DTT, 1 mM PMSF, 10,000 gels U/ml MNase). The reaction was stopped by the

addition of EDTA to a final concentration of 10 mM, when approximately 80% of genomic

DNA was reduced to mononucleosomes. The MNase-digested samples were then sonicated

and centrifuged at 16,000 × g for 10 min at 4°C to remove debris, and the resulting

supernatants were diluted 1:5 in ChIP dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM

NaCl, 1 mM EDTA, 1% Triton-X-100). This material was then used for

immunoprecipitation.

On day 1, each 2 μg of antibodies to YAP, TEAD1, or normal control rabbit IgG

were pre-bound to 50 μl of anti-rabbit or anti-mouse IgG magnetic beads (Invitrogen)

overnight at 4°C. On day 2, beads were washed three times with 5 mg/ml BSA/PBS, and

immunoprecipitation reactions were carried out with chromatin extracts overnight at 4°C.

Five per cent of the chromatin extract was set aside for input. On day 3, beads were washed

two times with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM

EDTA, 1% Triton-X-100), followed by twice with high salt wash buffer (20 mM Tris-HCl

pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton-X-100), once with LiCl wash buffer (250

mM LiCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate),

and once with TE buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA). Beads were then

resuspended in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and

incubated for 30 min at 65°C with frequent mixing. The resulting eluate and input samples

from day 1 were transferred into new tubes, and reverse crosslinking reactions were carried

out overnight at 65°C. On day 4, samples were treated with RNase A (0.2 mg/ml) for 1 h at

37°C, followed by proteinase K (0.2 mg/ml) digestion for 2 h at 50°C. DNA was then

ethanol-precipitated and purified using the PCR purification kit (Qiagen). Precipitated DNA

was quantitated by real-time PCR analysis. The primer sequences for ChIP analysis

(forward and reverse, respectively) are 5'-CTTTGGAGAGTTTCAAGAGCC-3' and 5'-

TCTGTCCACTGACATACATCC-3' for CTGF; 5'-TTGACTCACCCTGCCCTCAATATC-

3' and 5'-TTTCATTCCATCCAGCCTGGGG-3' for GAPDH; 5'-

TCCTTCTACGGATGCGGTTGACAG-3' and 5'-ATTCTTGCTTAGCGTTCTTTCCCC-3'

for LATS1; 5'-TGGTAACTTGGTGGCTCCTTCAGG-3' and 5'-

TTTGCCCTCTCGACTGCATTTG-3' for LATS2-1; 5'-

AATACAAATGCAGTCGAGAGGGC-3' and 5'-TCTCGGACCTATTTGACTGGCTG-3'

for LATS2-2; 5'-GCCATTTTCAACCTTTTAGCCCC-3' and 5'-

TCCTGCCATCCATCAAACAGCC-3' for LATS2-CR1; 5'-

TGTTCCGTGCTGATTTCCCTCTG-3' and 5'-ACCAAGGCTTTGTTTTCCCTGG-3' for

LATS2-CR2; 5'-CAGCAATTTCATAGGATGCCAGG-3' and 5'-

CCTCGTGAGGTTGTTGAATTTCAG-3' for NF2; 5'-

ACACAGCACGGACACACACGC-3' and 5'-TTCCCCACCTCAAACCCTGG-3' for

NF2-CR. All ChIP signals were normalized to the input (labeled as % of input on the

vertical axis).

Transwell cell migration assay

Cell migration assays were performed using BD FalconTM cell culture inserts for 24-well

plates with 8 μm pores filter, and the bottom filter was pre-coated with fibronectin (20

μg/ml). HEK293A cells infected with retrovirus encoding tetracycline-inducible Myc-

YAP(5SA) construct (or control) were cultured in the presence of doxycycline (200 ng/ml)

for 24 h and then were seeded (3 × 104 cells) into the upper chamber of the insert in serum-

free media with doxycycline. Lower chamber was filled with complete medium with

doxycycline. After 7 h, cells were fixed and stained with 0.1% crystal violet in methanol.

Cells in the upper chamber were carefully removed, and cells that migrated through the

filter were assessed by photography. For quantification, filters were removed from the

insert and cells were stained with DAPI, and nuclei were counted under a fluorescence

microscope.

YAP mutant mice

To achieve liver-specific or intestine-specific gene deletion, YapF/F mice (Zhang et al. 2010)

were bred to Albumin-Cre or Villin-Cre transgenic mice, respectively. Two- to three-month-

old sex-matched mice were used for all experimental procedures. For liver-specific YAP

transgenic mice, 2-month-old YAP transgenic and non-transgenic littermates were fed 0.2

mg/ml doxycycline (Sigma) in drinking water supplemented with 2.5% sucrose, as

described previously (Dong et al. 2007). Mice were euthanized 2 weeks after induction, and

the livers were harvested and immediately frozen in liquid nitrogen. Frozen tissues were

pulverized on dry ice, and powdered tissue samples were homogenized in RIPA buffer (20

mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM

PMSF, protease inhibitor cocktail, 400 μM Na3VO4, 400 μM EDTA, 10 mM NaF, and 10

mM sodium pyrophosphate) using a tissue tearer. Homogenates were then centrifuged at

16,000 × g for 10 min at 4°C twice to remove debris, and approximately 10 μg of the

resulting supernatant proteins were used for immunoblot analysis. Similarly, powdered

frozen tissue samples were subjected to RNA extraction. For immunohistochemical

staining, tissue samples were prepared as described previously (Zhang et al. 2010;

Taniguchi et al. 2015). Paraffin-embedded tissue sections were dehydrated followed by

antigen retrieval and endogenous peroxidase inactivation. Immunohistochemical staining

was performed with the use of Mouse on Mouse (M.O.M.™) Kits (Vector laboratories)

according to the manufacturer's protocol. TAZ primary antibody (1:100) was detected using

Vectastain elite ABC kit and DAB substrate kit (Vector Laboratories). Hematoxylin was

used for nuclear staining.

Statistical analysis

Quantitative data are presented as mean ± SD and were compared between groups with the

two-tailed Student’s t test as performed with Microsoft Excel software. A p value of <0.05

was considered statistically significant. For correlation analysis, gene expression data

across a panel of 967 cancer cell lines were downloaded from the Cancer Cell Line

Encyclopedia (CCLE) (Barretina et al. 2012), and the correlations between mRNA

expressions of each pair of genes were evaluated by Pearson’s correlation coefficient (r)

with two tailed p-values < 0.05 considered significant.

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