Supplemental Information Supplemental figure...

1

Supplemental Information

Supplemental figure legends

Figure S1. Supplemental figure for Fig. 1.

A. Different methods of cell detachment similarly induce YAP phosphorylation.

MCF10A cells were trypsinized and attached onto fibronectin-coated petri dishes

for 2 hours. Cells were either directly lysed (Attach) or detached with Trypsin-

EDTA, Trypsin alone, or EDTA alone. Samples were analyzed by western blots

with anti-YAP antibody.


A. Cytochalasin D blocks cell attachment-induced YAP dephosphorylation.

MCF10A cells were trypsinized (T) and attached onto fibronectin-coated petri

dishes for 1.5 hours (A). Inhibitors were added at the time of plating as indicated.

Cell lysates were resolved on Phos-tag-containing SDS-PAGE gels and western

blot was done as indicated.

B. Scr, FAK, ROCK, and microtubule inhibitors do not have significant effect on

attachment-induced YAP dephosphorylation. Experiments were similar to that in

A except different inhibitors were used.

C. Scr, FAK, and actomyosin inhibitors do not block cell detachment-induced YAP

phosphorylation. MCF10A cells were replated onto fibronectin-coated petri

dishes for 1.5 hours. During the last half an hour, indicated inhibitors were added

2

to the culture medium. Cells were then trypsinized and lysed. Same

concentrations of inhibitors were also included in trypsin.

D. Actomyosin and ROCK inhibitors do not induce YAP dephosphorylation in cells

cultured at high density. MCF10A cells cultured at high cell density were treated

with indicated inhibitors for one hour. Cells were then lysed and cell lysates were

resolved on Phos-tag-containing SDS-PAGE gels for western blot as indicated.

E. Nocodazole disrupts microtubule and induces actin stress fibers. Cells were the

same as these in Fig. 2F. Briefly, MCF10A cells were cultured onto fibronectin-

coated cover glasses at high cell density. Cells were treated with nocodazole

before fixation. Samples were then stained with anti-alpha-tubulin antibody for

microtubule and Alexa Fluor 488-Phalloidin for F-actin.


A. RNAi efficiency for Fig. 4A. Cell lysates were analyzed by western blots for the

expression of Lats1, Lats2, Mst1, and Mst2.

B. RNAi efficiency for Fig. 4B,C. Cell lysates were analyzed by western blots for

the expression of Lats1, Lats2, Mst1, and Mst2.

C. Specificity of phospho-Lats1/2 antibodies. HEK293 cells were transfected with

indicated plasmids. Lats2 was immunoprecipitated with anti-HA antibody and

then treated with lambda protein phosphatase as indicated. Immunoprecipitates

were analyzed by western blotting.

Figure S4. Mst1/2 activity is not regulated by cell attachment.

3

A. Cell attachment status does not regulate the activity of ectopically expressed

Mst2. Plasmids encoding Flag-Mst2 was transfected into HeLa cells. Cells were

freshly attached to fibronectin-coated petri dishes for 2 hours (A) or further

trypsinized (T). Mst2 was then immunoprecipitated by anti-Flag antibody and was

subjected to in vitro kinase assays using GST-Mob purified from E.coli as a

substrate in the presence of 32P-ATP. The products were analyzed by

autoradiograph.

B. Cell attachment status does not regulate the activity of endogenous Mst1.

Experiments were similar to that in A except that endogenous Mst1 was

immunoprecipitated from HeLa cells.

C. Mst2 phosphorylation is not regulated by cell attachment. Mst2 transfected HeLa

cells were subjected to attachment for 2 hours (A) or further trypsinization (T).

Mst2 phosphorylation was detected by a phosphoMst2 (T183)-specific antibody

in western blotting.

D. Mob phosphorylation is not regulated by cell attachment. MCF10A cells were

subjected to trypsinization (T), followed by attachment for 2 hours (A), and then

re-trypsinization (reT). Cell lysates were analyzed by western blotting for Mob

phosphorylation on T35, a known Mst1/2 target phosphorylation site. Cells

treated with hydrogen peroxide in a separate experiment was used a positive

control for Mob phosphorylation.

E. Knockout of Mst1 and Mst2 in MEF cells does not affect YAP phosphorylation

induced by cell detachment. Mst1 and Mst2 double knockout MEF cells were

generated by adenoviral infection of Mst1-/-, Mst2flox/- MEF cells. Ablation of

4

Mst1 and Mst2 was confirmed by western blots. Cells were then subjected to

trypsinization (T), followed by attachment for 2 hours (A), and then re-

trypsinization (reT). Cell lysates were resolved on Phos-tag-containing SDS-

PAGE gels and anti-YAP antibody was used for western blot analysis.

Figure S5. Cell attachment does not regulate interaction of Hippo pathway core

components.

A. Cell attachment status does not regulate Mst2-Sav interaction and Lats2-Mob

interaction. Indicated plasmids were co-transfected into HeLa cells. Cells were

freshly attached to fibronectin-coated petri dishes for 2 hours or further

trypsinized. Flag-tagged proteins were immunoprecipitated with anti-Flag

antibody and co-immunoprecipitation was analyzed by western blots with anti-

HA antibody.

B. Cell attachment status does not regulate the formation of the Hippo pathway core

components complex. Indicated plasmids were co-transfected into HeLa cells.

Cells were freshly attached to fibronectin-coated petri dishes for 2 hours or further

trypsinized. Flag-Lats2 was immunoprecipitated with anti-Flag antibody and co-

immunoprecipitation of other proteins were analyzed by western blots with anti-

HA antibody.

Figure S6. Supplemental figure for Fig. 5 and Fig. 6.

5

A. YAP induces Erk activation and EMT in detached cells. Cells the same as these in

Fig. 5C were harvested and lysed for western blot analysis using indicated

antibodies.

B. Lats1/2 and YAP/TAZ knockdown efficiency in HMLE, RWPE, and ACHN

cells. Cells the same as those in Fig. 5D (top panel), Fig. 6A (middle panel), and

Fig. 6B (bottom panel) were analyzed for knockdown efficiency of indicated

proteins by western blots.

C. YAP/TAZ knockdown does not induce anoikis in PC-3 cells. PC-3 cells were

transfected with control siRNA or siRNAs targeting YAP and TAZ. Cells were

then cultured on tissue culture plates (Attach) or ultra-low attachment plates in

suspension. After 72 hours, cells were collected and stained with Annexin V-PE

and analyzed by FACS.

D. YAP/TAZ knockdown has only minor effect on inducing anoikis in DU 145 cells.

Experiments were similar to that in panel C except that DU 145 cells were used.

E. YAP/TAZ knockdown promotes anoikis in SF268 cells. Experiments were similar

to that in panel C except that SF268 cells were used.

F. Histogram showing the expression value of Lats1 and Lats2 in a cohort of

samples including benign adjacent tissue, localized (PCa) and metastatic (Met)

prostate tumors. P value measures the statistical difference between metastatic

prostate cancer when compared to benign and localized prostate cancer.

Zhao_FigS1.

YAP(Phos-tag)

Attac

hTry

p-EDT

A

Tryp

Enzy

me Fr

eeA

Zhao_FigS2.

BA

C

YAP(Phos-tag)

AT AAA10 50

Blebb

istati

n (µM

)

Cyto

D (0.

5 µM)

YAP(Phos-tag)

T A AA A AA A APP

2 5µM

PF27

1 1µM

Lat B

1µg/m

l

Noco

1µM

Y276

32 10

µM

Taxo

l 5µM

Vinbla

stin 5

µM

YAP(Phos-tag)

PP2 (µM)

A T T T T T T T1 10 20 1 5 20

PF27

1 (µM

)

Blebb

istati

n (µM

)

Blebb

istati

n 20µ

M

Y276

32 10

µM

H115

2 10µ

M

-

D

YAP(Phos-tag)

Noco(1μM, 1h)

Control

F-actinTubulinE

Zhao_FigS3.

BA

Tubulin

Lats1

Lats2

Mst1

Mst2

Contr

ol

Lats1

+Lats

2

Mst1+

Mst2

Tubulin

Lats1

Lats2

Mst1

A TA TA TsiRNA Co

ntrol

Lats1

+ Lats

2

Mst1+

Mst2

Contr

ol

Lats1

+ Lats

2

Mst1+

Mst2

Mst2

HA

pLats (T999)

pLats (S872)

HA-Lats2+ +

S872

AW

TW

TW

T

Flag-Mst2 - -Lambda PPase - +- -

C

siRNA

C

Zhao_FigS4.

BA

D

Mst1

YAP(phos-tag)

32P-Mob

32P-Mst1

GST-Mob IP

lysate

A T A T

IP C. IgG Mst1

GST-Mob

Flag-Mst2 WT KR -A

WT KR -T

32P-Mob

32P-Mst2

32P-Mob

(L)

(S)

Flag-Mst2

Flag-Mst2

pMst2 (T183)TA

Flag-Mst2 + +

YAP(Phos-tag)

T reTA T reTA

Mst1-/-Mst2cre/-

Mst1+/+Mst2+/+

Hsp90

Mst1

Mst2

Mst1-/-Mst2cre/-

Mst1+/+Mst2+/+

E

pMob1 (T35) (S)

pMob1 (T35) (L)

Mob1

H2O2- T reTA

Zhao_FigS5.

BA

Flag-Lats2 - +

A T A T A T A T

-----

Flag-Mst2HA-Sav

HA-Mob- -- ---

-- --

-- --+

+++++

+++++

IP:Flag

Lysate

WB: HA

WB: Flag

WB: HA

WB: Flag

IP:Flag

Lysate

WB: HA

WB: Flag

WB: HA

WB: Flag

Flag-Lats2 -T A T A

-

HA-Mst2HA-Mob

HA-Sav

++++

++++++++

++

Zhao_FigS6.

CA

Attach

Suspension

Non-targeting siRNA YAP/TAZ siRNA

6.9% 8.6%

12.3% 22.5%

ED

Attach

Suspension


2.5% 1.5%

3.3% 4.2%

Attach

Suspension


8.9% 5.9%

14.4% 17.6%

pERK

ERK

E-cad

N-cad

Tubulin

Myc-YAP - WTA S

5SA - WT 5SA

F

-3 -2.5

-2 -1.5

-1 -0.5

0 0.5

1 1.5

2 2.5

1 6 11

16

21

26

31

36

41

46

51

56

61

66

71

76

81

86

91

96

101

106

111

116

121

126

131

136

141

146

151

benign PCa Met

-5

-4

-3

-2

-1

0

1

2

1 6 11

16

21

26

31

36

41

46

51

56

61

66

71

76

81

86

91

96

101

106

111

116

121

126

131

136

141

146

151

benign PCa Met

Lats1

Lats2

P value

0.01

3.28E-06

B

P valueLats1+Lats2

-5

-4

-3

-2

-1

0

1

2

3

4

1 6 11

16

21

26

31

36

41

46

51

56

61

66

71

76

81

86

91

96

101

106

111

116

121

126

131

136

141

146

151

benign PCa Met

2.30E-08

Tubulin

TAZ

YAP

Tubulin

Lats1

Lats2

siYAP/TAZ - + - +A S

siLats1/2 - + - +A S

Tubulin

Lats1

Lats2

siLats1/2 - + - +A S

Supplemental Information11-5-11Supplemental figuresFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6

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