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24

Figure Legends

FIGURE 1. Mob1a/1b deletion in mouse SMG epithelium results in significant acinar

cell hypoplasia and immature ductal cell hyperplasia.

(a) Left: Representative macroscopic views of SMGs of 8–9-week old control and

adMOB1DKO mice 3 weeks after starting TAM treatment for 5 days (post-TAM). Scale

bar, 0.5 cm. Middle: Quantitation of SMG weight at the indicated days post-TAM (n=37/group). Right: Total cell number of each SMG at 21 days post-TAM (n=7/group) was

determined by counting cells after digestion with collagenase, hyaluronidase, and dispase

as described in “Experimental Procedures.” For all Figures, data are the mean ± SEM.

**P<0.01; *P<0.05. (b) Upper left: H&E-stained sections of control and adMOB1DKO

SMG at 21 days post-TAM. Scale bar, 10 μm. Upper middle and right: Representative

immunohistochemical analyses of AQP5 (upper middle; green) or CK7 (upper right; red)

expression in control and adMOB1DKO SMGs at 21 days post-TAM (n=6/group). Nuclei

were stained with DAPI (blue). Scale bars, 20 μm. Lower: Quantitation of percentages of

AQP5+ or CK7+ cells (left, middle) or cell number (right) of control and adMOB1DKO

SMGs at 21 days post-TAM (n=7/group). The cell numbers were calculated from total

cell number of each SMG multiplied by the percentage of AQP5+ and CK7+ cells,

respectively. (c) Upper: Representative immunohistochemical staining to detect CK14CK7+ mature ductal cells and CK14+CK7+ immature ductal cells in SMGs of control and

adMOB1DKO mice at 21 days post-TAM (n=6/group). Scale bar, 20 μm. Lower:

Quantitation of total cell numbers of the immature (left) and mature (right) ductal cells in

the SMGs in the upper panel. (d) H&E-stained sections of control and adMOB1DKO

SMGs at 21 days post-TAM showing dysplastic enlarged nuclei in the mutant tissue.

Scale bar, 10 μm.

FIGURE 2. adMOB1DKO mice show impaired saliva production, accumulating

inflammatory cells, and fibrosis similar to Sjögren’s syndrome.

(a) Quantitation of the total amount of saliva secreted in 60 min by control and

adMOB1DKO mice subjected to Pilocarpine stimulation in vivo (n=4/group). (b)

Representative sections of control and adMOB1DKO SMGs at 14 days post-TAM that

were stained with H&E, anti-CD45 antibody, or Sirius Red. Scale bars, 20 μm. Nuclei

25

were counterstained with hematoxylin (top panels), DAPI (middle panels), and iron

hematoxylin (bottom panels). Blue arrowheads, inflammatory cells; yellow arrowheads,

fibroblasts.

FIGURE 3. MOB1 deficiency has no effect on the proliferation or apoptosis of acinar

cells but ductal cells show increased turnover.

(a) Representative immunohistochemical images (left) and quantitation (right) of PCNA+

cells (green) among CK7+ ductal cells (red), AQP5+ acinar cells (red), or total cells in

control and adMOB1DKO SMGs at 21 days post-TAM (n=6/group). (b) Representative

immunohistochemical images (left) and quantitation (right) of TUNEL+ cells (red) among

CK7+ ductal cells (green), AQP5+ acinar cells (green), or total cells in control and

adMOB1DKOSMGs at 21 days post-TAM (n=6/group). For (a) and (b), nuclei were

stained with DAPI. Scale bars, 10 μm.

FIGURE 4. MOB1-deficient immortalized clonal salivary epithelial cells show

impaired acinar cell differentiation, and MOB1-deficient SMGs contain acinar/ductal

bi-lineage progenitors.

(a) Quantitation of the fold increase in the expression of the indicated acinar (Aqp5 and

Amy1a) and ductal (Krt19 and Krt7) lineage markers as determined by qPCR in

imMOB1DKO cells before (-) and after (+) culture in Matrigel for 7 days (n=4-5/group).

mRNA expression data were normalized to Gapdh. (b) Quantitation of the fold increase

in expression of the indicated acinar and ductal lineage markers as determined by qPCR

in imMOB1DKO cells that were cultured for 10 days in Matrigel with (+) or without (-)

TAM (n=4-5/group). mRNA expression data were normalized to Gapdh. Mob1a as

inhibitory control after TAM. (c) Representative immunofluorescent detection (left) and

quantification (right) of AQP5+CK14+ (double positive) immature ductal cells (white

arrowhead) among total CK14+ ductal cells in control and adMOB1DKO SMGs at 21

days post-TAM (n=6/group). Scale bar, 10 μm.

FIGURE 5. MOB1-mediated regulation of TAZ rather than YAP1 controls adult

salivary epithelial cell homeostasis.

(a) Immunoblot to detect total TAZ and YAP in total extracts of control and

26

adMOB1DKO SMGs at 21 days post-TAM. Actin, loading control. (b) Immunostaining

to detect total TAZ and YAP1 in control and adMOB1DKO SMGs at 21 days post-TAM.

Scale bar, 20 μm. For (a) and (b), data are representative of three independent

experiments. (c) Left: Representative H&E-stained sections of SMGs from control,

adMOB1DKO, adTAZTKO and adYAPTKO mice at 21 days post TAM (n=5/group).

White arrowheads, acinar cells. Scale bar, 20 μm. Right: Quantitation of the percentages

of acinar cells among total cells of the SMGs in the left panel (n=5/group).

FIGURE 6. MOB1-deficient salivary epithelial cells show inactivation of SOX2 and

SOX10, but activation of β-catenin.

(a, b) Representative immunostaining (left) and quantification of the intensities of nuclear

SOX2, SOX10 (a) and active β-catenin (b), in SMGs of control and adMOB1DKO mice

at 21 days post-TAM (n=5/group). Represents are relative intensities of these molecules

in adMOB1DKO mice to those of control mice. Scale bars, 20 μm.

27

80

**

40

AQP5

AQP5+

adMOB1

DKO

CK7+

DKO

cont

**

CK7

cont

(d)

0.5

**

0.25

cont

DKO

cont

(c)

cont

d21

CK14- CK7+

Cell number (x106)

d14

d21

DKO

cont

CK14+ CK7+

Cell number (x106)

d10

**

DKO

40

Total Cell number (x106)

cont

adMob1DKO

cont

DKO

H&E

Cell number (x106)

d7

cont

**

80

DKO

80

cont

40

Weight of SMG (mg)

adMOB1

DKO

CK7+ (%)

(b)

DKO

cont

cont

AQP5+ (%)

(a)

adMOB1

DKO

CK14

CK7

0.5

adMOB1

DKO

**

FIGURE 1

(b)

Secretion of saliva (µL/h )

(a)

cont

adMOB1

DKO

H&E

300

200

100

CD45

cont

adMOB1

DKO

Sirius Red

FIGURE 2

(a)

cont

adMOB1

DKO

PCNA+ (%)

PCNA

AQP5

PCNA

CK7

cont

adMOB1DKO

**

CK7+

total

cont

adMOB1DKO

**

AQP5+

(b)

cont

adMOB1

DKO

Tunel+ (%)

TUNEL

AQP5

TUNEL

CK7

**

AQP5+

CK7+

total

FIGURE 3

(b)

(c)

Aqp5

AQP5

cont

adMOB1

DKO

**

Krt7

CK14

**

Amy1a Krt19

**

**

Aqp5 Amy1a Krt19

Krt7

**

Mob1a

CK14 AQP5

DKO

**

- TAM

+ TAM

cont

**

Fold increase

Fold increase

- Matrigel

+ Matrigel

CK14+ AQP5+ / total CK14+ (%)

(a)

Figure 4

(a)

(b)

adMOB1

DKO

cont

adMOB1

cont

DKO

MOB1

TAZ

TAZ

YAP

Actin

YAP

(c)

80

**

**

**

**

40

adYAP1 TKO

adTAZ TKO

adYAP1 TKO

adMOB1 DKO

adTAZ TKO

cont

adMOB1 DKO

Acinar cell (%)

cont

FIGURE 5

(a)

(b)

SOX2

Active b-catenin

SOX10

cont

cont

Relative SOX2

Intensity

**

0.5

cont

DKO

0.5

cont

DKO

Relative Active

b-catenin Intensity

adMOB1

DKO

Relative SOX10

Intensity

adMOB1

DKO

1.5

0.5

cont

DKO

FIGURE 6

Supplemental Information

TAZ inhibits acinar cell differentiation but promotes immature ductal cell

proliferation in adult mouse salivary glands

Yosuke Miyachi1,6, Miki Nishio1,6, Junji Otani1, Shinji Matsumoto2, Akira Kikuchi2, Tak

Wah Mak3,4,5, Tomohiko Maehama1,7,8 and Akira Suzuki1,7,8

Division of Molecular and Cellular Biology, Kobe University Graduate School of

Medicine, Kobe, Japan

Department of Molecular Biology and Biochemistry, Graduate School of Medicine,

Osaka University, Suita, Japan

The Princess Margaret Cancer Centre, University Health Network, Toronto, Canada

Departments of Immunology and Medical Biophysics, University of Toronto, Toronto,

Canada

Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong,

Hong Kong, SAR

Equal contribution as first author.

Equal contribution as last author.

Corresponding authors:

Tomohiko Maehama, Division of Molecular and Cellular Biology, Kobe University

Graduate School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe, Hyogo, 650-0017,

Japan

Tel:+81-78-382-6052; Fax:+81-78-382-6053; E-mail: tmaehama@med.kobe-u.ac.jp

Akira Suzuki, Division of Molecular and Cellular Biology, Kobe University Graduate

School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe, Hyogo, 650-0017, Japan.

Tel:+81-78-382-6051; Fax:+81-78-382-6053; E-mail: suzuki@med.kobe-u.ac.jp

Supplementary Table

Table S1. List of primer sequences for genotyping and qPCR.

Supplementary Figure Legends

Fig. S1. Protocol to generate postnatal Mob1a/1b DKO mice (adMOB1DKO) and their

controls.

(a) Diagram of the protocol used to generate adMOB1DKO mice. Mob1aflox/flox;Mob1b−/−

mice

were

crossed

to

Rosa26-CreERT2-Tg

mice

to

produce

Rosa26-

CreERT2;Mob1aflox/flox;Mob1b−/− and control Mob1aflox/flox;Mob1b−/− progeny. When the

progeny were 35-42 days old, tamoxifen (TAM) was i.p. administered for 5 days. Mice

were then sacrificed on the indicated days post-TAM initiation. (b) H&E-stained sections

of SMGs from mice of the indicated genotypes at 3 weeks after starting TAM application

(+TAM) or not (-TAM). Scale bar, 20 μm. Blue arrowheads, acinar cells; yellow

arrowheads, ductal cells. Results shown are representative of at least three independent

trials.

Fig. S2. Confirmation of gene deletion in adMOB1DKO mice.

Representative immunofluorescent staining to visualize target DNA (Mob1a) in SMGs of

Rosa26-CreERT2; Mob1aflox/flox;Mob1b-/- mice that expressed the Rosa26-LSL-tdTomato

transgene and were treated with (+) or without (-) TAM i.p. for 5 days. Sections were

stained with anti-RFP antibody to detect tdTomato expression. Scale bar, 20 μm. Mob1a

deletion was observed in most of the cells in the mutant SMG.

Fig. S3. Confirmation of deletion of TAZ or YAP1 in triple knockout (TKO)

MOB1A/1B-deficient mice.

Representative immunohistochemical analyses to detect total TAZ or YAP1 in SMGs of

control (left), adMOB1DKO (middle), and MOB1A/1B & TAZ TKO (adTAZ TKO;

Rosa26-CreERT2;Mob1aflox/flox;Mob1b−/−;Tazflox/flox;+TAM) (right, top); or MOB1A/1B

YAP

TKO

(adYAP1

TKO;

Rosa26-

CreERT2;Mob1aflox/flox;Mob1b−/−;Yap1flox/flox;+TAM) (right, bottom) mice. TAZ or YAP1

expression was deleted in most of the cells in SMGs of adTAZ TKO or adYAP1 TKO

mice, respectively. Scale bar, 20 μm.

Fig. S4. MOB1-deficiency did not alter GLI2 expression in salivary epithelial cells

Representative immunostaining (left) and quantification of GLI2 (a) in SMGs of control

and adMOB1DKO mice at 21 days post-TAM (n=5/group). Scale bars, 20 μm.

Fig. S5. TAZ is activated by duct ligation.

(a) Diagram of the protocol to achieve salivary duct ligation in mice. Control mice (5week old) were subjected (or not) to duct ligation and sacrificed 1 week later. (b)

Immunoblot to detect TAZ in SMGs of control (cont) and duct-ligation (DL) operated.

Data are representative of three independent experiments. Actin, loading control. (c) H&E

staining (upper) and immunostaining to detect TAZ (lower) in SMGs of the mice in (b).

(n=3/group). Scale bars, 20 μm.

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