2016-03-04 supplemental figure.ppt [兼容模式]n-terminal rankl rankl c-terminal n-terminal...

Figure S1

c d

RANK

IGG

RANKLIGG

IP: FLAGIB: FLAG

IP: FLAGIB: RANKL

a

IB FLAG

LGR4 NTLRR8

130100

170

35425572

M（KD）

25

17

130100

35425572

25

17

170

M（KD）

LGR4 NTLRR14

b

-100 0 100 200 300(s)

0

Res

pons

e U

nit (

RU

)

100

200

-100

400 nM200 nM100 nM50 nM25 nM

e

LGR4ECD Regeneration OPGFc LGR4ECD Regeneration

Res

pons

e U

nit (

RU

)

100

150

200

250

300

50

Bind: 46.2 RUStability: 54.2RU



f gLGR4 ECD LGR4 ECD

RANKL RANKLN-terminalC-terminal N-terminal

C-terminal

Nature Medicine: doi:10.1038/nm.4076

Figure S2

b c dIgG Ctrl silgr4

0

120

Cou

nts

100 104102

FL1-H

Ctrl IgG

0

0

120

Cou

nts

100 104102

FL1-H

a

20 200

Ctrl IGG

Vector

0

80

Cou

nts

100 104102

FL1-H

LGR4

IP：FLAGIB：FLAG

IP：FLAGIB：RANK

IB：FLAG

IB：RANK

IP：FLAGIB：FLAG

IP：FLAGIB：RANK

IB：FLAG

IB：RANK

IGG

LGR4FLAG

RANK+++

-

LGR4FLAG

RANK+++

-

RANKL- - ++RANKL f

0

40

80

120

Ctrl IgG 0 20 200

LGR4 ECD (ng/ml)

0

40

80

120

Fflu

ores

cenc

e In

tens

ity (

% V

ecto

r)

0

40

80

120

160

Fflu

ores

cenc

eIn

tens

ity (

% C

trl)

Fflu

ores

cenc

e In

tens

ity (

% R

AN

KL)

e

IB RANKL

IB His

IP FLAGIB RANKL

LGR4-ECDFLAG

RANKL

RSPO1His

+ + + + ++ + + +

IP FLAGIB HisIP FLAGIB FLAG

IGGRANKL


0

40

80

120

160

0 20 100 200 500 1000 2000cAM

P (

pmol

/106

cells

)LGR4Vector

0 100 200

CR

E-L

uc(f

old)

0

0.4

LGR4Vector

0.8

1.2

1.6

2.0

NFA

T-Lu

c(fo

ld)

0

20

40

60

0 100 200

LGR4

Vector

SR

F-R

E L

uc(f

old)

0

1

2

0 100 200 20%FBS

3

a b

PMA+IONO

LGR4

Vector

RANKL(ng/ml)

RANKL(ng/ml)

RANKL(ng/ml)

RANKL(ng/ml)

Figure S3

c

200100

RANKL(ng/ml)

Cal

cium

res

pons

e (f

old)

0

0.2

0.4

0.6 LGR4

Vector


Figure S4

Gapdh

Ctsk

Acp5

Calcr

Nfatc1

Lgr4

Src

Lgr5

Lgr7

Lgr8

Tshr (Lgr3)

Lhcgr (Lgr2)

Fshr (Lgr1)

Lgr6

a

b

TRAP+LacZ+

LacZ+

Lgr4

Lgr4

Lgr4

c-Src

Gapdh

Ctsk

Acp5

Calcr

Nfatc1

0 2 3 4 5 6 day

c

Input

Lgr4

Rank

Gapdh

M-CSF(10ng/ml)

0 2 31

d

Lgr4

Gapdh

RANKL

CsA

－

－－

+ +

+

e

Ctsk

c-Src

Acp5

Nfatc1

Gapdh

Lgr4

CA-NFATc1

fLg

r4 e

xpre

ssio

n (f

old)

0

1

2

3

RANKL

CsA

－

－－

+ +

+

Lgr4

expr

essi

on (

fold

)

0

2

4

6

CA-NFATc1

dH2O

Inpu

t

IgG

NFA

T

RA

NK

L

NT

BS

2

NT

BS

3

N

TB

S1

g


Figure S5

a

Lgr4 locus

Targeting vector6 7 8 9 10 11 1213 14 15 16 17 18

8 9 10 11 1213 14 15 16 17 18

Targeted allele after Neo deletion

8 9 10 11 1213 14 15 16 17 18

Targeting allele after Cre recombination

8 9 10 11 1213 18

loxP FRT Neo Exon Deleted Exon

b

Lgr4

fl/fl

Lgr4

CK

O

0

0.1

0.2

0.3

Tb.

Th

(m

)

Tb.

N (

1/m

m)

Tb.

Sp

(m

)

BV

/TV

(%

)

0

10

20

40

80120

0

0

0.4

0

200

400

600

800

** **

*

*

0.8

1.2

1.6

Lgr4 CKOLgr4fl/fl

30 160

Tra

becu

lar

bone

B

MD

(g/

cm3 )


b

Gapdh

Acp5Calcr

Src

Nfatc1

0 3 4 0 3 4 Day

Ctsk

WT Lgr4

Figure S6

0 5 10025

WT

Lgr4

a

Ctr

lsi

Lgr4

Vec

tor

LGR

4

TR

AP

+ce

lls (

% o

f Ctr

l)

40

160

80

120

**

0

TR

AP

+ce

lls (

% o

f Vec

tor)

0

40

80

120 **

c

d

RN

Ai e

ffica

cy o

f Lgr

4 (%

of C

trl)

0

40

80

120

Ove

rexp

ress

ion

LGR

4 (%

of V

ecto

r)

0

400

800

1,200

1,600

RANKL (ng/ml)


Figure S7 a

d

b

e

0

100

200

TR

AP

cells

50ng/ml 100ng/mlCtrl RANKL

c

0

200

400

TR

AP

cells

RSPO-1

RANKL

(ng/ml)

50 100

RSPO150ng/ml

RSPO1100ng/ml

pp65

IB

GAPDH

p65

WT Lgr4

min RANKL0 5 10 15 30 600 5 10 15 30 60

Vector CA-Gαq

NFATc1

Histone3

0 4 8 122 0 4 8 122 hour RANKL

NE

NFATc1

Histone3

CE Gαq

βactin

Vector RGS2

0 30 6015 0 30 6015 min RANKL

GSK3

GαqCT

0 3015 60p-GSK3

(Ser9)

Rankflox/flox; AdCre

0 15 30 min RANKL

GSK3

60 90 0 15 30 60 90

pGSK3(Ser9)

Rankflox/flox; AdGFPf

g

0 100 500 2,000

TR

AP

pos

itive

cel

ls

0 100 5002,000

LGR4ECD (ng/mL)

0

40

80

120

160***

**LGR4ECD (ng/ml)

h

0 20 50 100

TR

AP

cells

0

40

80

120

0 5020 100

**

*

LGR4ECD (ng/ml)

LGR4ECD (ng/ml)


Oc.

S/B

S (

%)

0

100

200

N.O

c/B

.Pm

0

10

20

30

ES

/BS

(%

)0

20

60

40

*** *** ***

300

400 40

Ctrl ECD Ctrl ECD Ctrl ECD

a bCtrl LGR4ECD

Veh

icle

RA

NK

L

Ctrl LGR4ECD

Veh

icle

RA

NK

L

Ctrl ECD Ctrl ECDVehicle RANKL

0

4

8

12

Bon

e vo

lum

e

（×1

06m

3

）

Ctrl ECD Ctrl ECDVehicle RANKL

0100200

TR

AP

area

(%

con

of c

on)

300400500 *** ***

* **

c

Ctrl LGR4ECD

Opg

0T

b.T

h (

m)

Tb.

N (

1/m

m)

Tb.

Sp

(m

)

BV

/TV

(%)

0

10

20

40

80

120

0

1

0

250

* *

2

330

* 500

Ctrl CtrlECD ECD Ctrl ECD Ctrl ECD

d

BM

D(g

/cm

3 )

0

0.1

0.2

0.3

0.4*

0

40

80

120

**

Ctrl LGR4ECD

Opg

TR

AP

area

(%co

n of

con

)

Ctrl ECD

Ctrl ECD

g

Figure S8

e

f

BV

/TV

(%

)

h


Histomorphometric parameters of osteoclast in calvaria bone

ParametersWT Lgr4-/-

8W

Oc.S/BS

N.Oc/B.Pm

Oc.Size

ES/BS

9.7±5.6 52.3±8.4

WT

16W

WT

24W

7.8±3.7 92.9±5.6 7.3±2.8 27.3±3.8

66.9±25.6 202.7±6.2

1.4±0.3 2.6±0.3

5.1±3.6 24.0±3.5

61.3±22.8 270.1±14.1

1.2±0.1 3.4±0.1

4.7±2.3 20.1±1.5

71.1±5.1 204.7±6.8

2.6±0.2 7.5±0.3

5.8±1.1 21.6±2.6

*

*

*

*

*

***

**

****

**

***

Lgr4-/- Lgr4-/-

ParametersWT Lgr4-/-

8W

Oc.S/BS

N.Oc/B.Pm

Oc.Size

ES/BS

23.7±6.0 79.6±15.5

WT Lgr4-/-

16W

WT Lgr4-/-

24W

113.7±12.5 178.2±9.4

2.1±0.3 4.5±0.6

4.0±0.4 24.4±1.7

24.4±3.0 64.6±5.8

100.8±2.3 162.6±2.8

2.4±0.2 4.0±0.4

5.0±1.5 26.0±3.3

8.8±2.2 55.0±2.2

56.9±1.7166.9±8.0

1.8±0.2 3.3±0.3

4.0±3.2 21.5±2.8

*

*

*

**

**

***

*

**

* *

**

*

*

Histomorphometric parameters of osteoclast in femur bone

Table S1


Table S2. Information of PCR primers

Gene Sequence Length

Nfatc1 sense 5' CCCGTCACATTCTGGTCCAT 3' 145bp antisense 5' CAAGTAACCGTGTAGCTGCACAA 3'

Src sense 5' TCCTTTGGGATTCTGCTGAC 3' 159bp antisense 5' CCAGCACTGGCACATAAGG 3'

Ctsk sense 5' ATGTGGGTGTTCAAGTTTCTGC 3' 505bp antisense 5' CCACAAGATTCTGGGGACTC 3'

Acp5 sense 5' CAGCTCCCTAGAAGATGGATTCAT 3' 70bpantisense 5' GTCAGGAGTGGGAGCCATATG 3'

Calcr sense 5' TGCAGACAACTCTTGGTTGG 3' 194bp antisense 5' TCGGTTTCTTCTCCTCTGGA 3'

Rank sense 5' CTGCTCCTCTTCATCTCTGTG 3' 244bp antisense 5' CTTCTGGAACCATCTTCTCCTC 3'

Fshr sense 5' TGCCCAACCATGGCTTAGA 3' 432bp antisense 5' TGATGGCCAGGATGCTGATA 3'

Lhcgr sense 5' ACCCGGTGCTTTTACAAACC 3' 407bp antisense 5' TGGCGATGAGCGTCTGAAT 3'

Tshr sense 5' TCATTGCCTCTGTAGACCTG 3' 119bp antisense 5' TGATAACTCACTGGCGAAA 3'

Lgr4 sense 5' AAGATAACAGCCCCCAAGAC 3' 260bpantisense 5' AGGCAGTGATGAACAAGACG 3'

Lgr5 sense 5' TCATCCAATCTCCTGTCGTC 3' 235bpantisense 5' TATGAAGGTCGTCCACACTG 3'

Lgr6 sense 5' ACATAACAACCGCATCCAGC 3' 251bpantisense 5' ATGCTGACCTTCCCACAAAC 3'

Lgr7 sense 5' GAAGCAAGTGAAGAAGGAGG 3' 249bpantisense 5' CTGTAGTTGTGCCAGAGTTG 3'

Lgr8 sense 5' ACGAACTCCACCTTCCTAAC 3' 209bpantisense 5' AGAAGAGGGTTGGATGACAG 3'

Gapdh sense 5' ACCCAGAAGACTGTGGATGG 3' 125bpantisense 5' TTCAGCTCAGGGATGACCTT 3'

Prdm1 sense 5′ TGCTTATCCCAGCACCCC 3′ 311bpantisense 5′ CTTCAGGTTGGAGAGCTGACC 3′

Maf b sense 5′ AACGGTAGTGTGGAGGAC 3′ 363bpantisense 5′ TCACAGAAAGAACTCAGGA 3′

NTBS1 sense 5‘ AGCAGAGCAATAGCAACAGC 3' 199bpantisense 5' CTCTGTTTCGCAAGTGCTTC 3'

NTBS2 sense 5' GAGTCCTTCCAGCTGTGTTA 3' 265bpantisense 5' GAGCTGGGGTTACACATTAC 3'

NTBS3 sense 5' CCACTCTGAATGCTGGATAG 3' 296bpantisense 5' CTTAGGGTGCTCAGCTTTAC 3‘

Lgr4 flox sense 5' CATCGAGATACTGGTCCTCAGCCA 3' 327bp (WT)antisense 5’TGACAAGACAGGCACTCCACAGCA 3’ 439bp (flox)

Lysm-cre MU 5' CCCAGAAATGCCAGATTACG 3' 350bp (WT)Com 5' CTTGGGCTGCCAGAATTTCTC 3' 700bp (MU)WT 5' TTACAGTCGGCCAGGCTGAC 3'


Supplementary Figure Legends:

Supplementary Figure 1. LGR4 interacts with RANKL (a)

Co–immunoprecipitation (IP) between RANK and RANKL as a positive control

of Fig. 1b. (b) Surface plasmon resonance (SPR) assay between OPG–Fc

protein and RANKL protein as a positive control of Fig. 1f-h. The KD between

RANKL and OPG–Fc is 9.03 nM. (c) SDS–PAGE of NT–LRR8 and NT–LRR14

proteins purified from HEK293T cell supernatant. NT–LRR8 and NT–LRR14

sequences were cloned into pcDNA4T0 plasmids and transfected into

HEK293T cells. Serum free cell culture medium was collected after 36 hours

and the proteins were purified using Ni–NTA magnetic beads. (d) The

NT–LRR8 and NT–LRR14 proteins were confirmed by immunoblotting using

the FLAG antibody. (e) SPR assay for competition analysis using the manual

run program. RANKL was immobilized CM5 sensor chip and the interaction

between LGR4–ECD and RANKL was markedly decreased from 46.2 RU to

16.0 RU when OPG–Fc protein was loaded onto the chip. (f, g) Molecular

modeling of RANKL and LGR4–ECD. The side view of the

RANKL/LGR4–ECD complex in a surface representation showed that

LGR4–ECD associated with RANKL in its concave surface. LGR4–ECD and

RANKL are colored in green and orange, respectively. The predicated

interaction region of LGR4 is highlighted in yellow (f). Ribbon representation of

the RANKL/LGR4–ECD complex showing details of the binding sites between


LGR4–ECD and RANKL. The predicated binding sites in LGR4 and RANKL

are cyan and magenta sticks, respectively (g).

Supplementary Figure 2. LGR4 interacts with RANKL (a) Co–IP analysis of

RANKL and LGR4–ECD association in HEK 293T cells treated with a dose

gradient of RSPO–1. Images are representative of 2 experiments with

biological replicate. (b) FACS analysis of RANKL binding to HEK293T cells

transfected with empty vector or LGR4 expression plasmids. RANKL

increased from 100% (control cells) to 143.54% (LGR4 overexpressing cells).

(c) FACS analysis of RANKL binding to HEK293T cells transfected with control

siRNA or LGR4–targeting siRNA as indicated. Knockdown of endogenous

LGR4 in HEK293T cells decreased RANKL binding from 100% (control cells)

to 60.37% (LGR4 knockdown cells). (d) FACS analysis of RANKL binding to

LGR4 positive HEK293T cells treated with indicated concentrations of

LGR4–ECD. The binding between RANKL (200 ng/mL) and LGR4 positive

HEK293T cells declined from 100% (0 ng/ml LGR4–ECD) to 72.53% (20 ng/ml

LGR4–ECD) or 65.45% (200 ng/ml LGR4–ECD). (e) Co–IP between LGR4

and RANK. Images are representative of 2 experiments with biological

replicate. (f) Co–IP between LGR4 and RANK following RANKL stimulation

for 20 min. Images are representative of 2 experiments with biological

replicate.


Supplementary Figure 3. RANKL has little effect on CRE–Luc, NFAT–Luc

or SRF–RE–Luc, or on cAMP production by LGR4. (a) CRE–, SRF–RE–,

NFAT– luciferase reporter gene expression in HEK293T cells treated as

indicated. Error bars, mean ± s.d; n = 3 per group. (b) RANKL has little effect

on cAMP production in HEK293T cells with or without LGR4 overexpression.

Error bars, mean ± s.d; n = 2 per group. (c) FLIPR calcium assay of

HEK293T cells transfected with empty vector or LGR4 expression plasmids

and treated with indicated concentration of RANKL. Error bars, mean ± s.d; n =

3 per group.

Supplementary Figure 4. Lgr4 was expressed in osteoclasts and Lgr4 is a

direct transcriptional target of RANKL/NFATc1 signaling. (a) PCR

reaction of lysates from BMMs prepared from 6–week–old C57BL/6 mice and

stimulated with 100 ng/mL RANKL and 10 ng/mL M–CSF for indicated times.

(b) Representative images (n = 3 images taken in total, one image from 3

different mice each) of LacZ/TRAP co–staining results of bone sections from 3

mice. Blue: LacZ staining represented for Lgr4; purple: TRAP staining. Scale

bar, 10 μm. (c) RT–PCR analysis to detect the expression of Lgr4 in BMMs

stimulated with RANKL in the presence of M–CSF. (d) RT–PCR analysis to

detect the expression of Lgr4 in BMMs treated with M–CSF alone. (e) Lgr4

expression in RAW264.7 cells stimulated with RANKL for 24 hours and treated

with vehicle or the calcineurin inhibitor cyclosporin A (CsA; 0.8 μM). RT–PCR


(top) and Real–time PCR (bottom). (f) Lgr4 expression in RAW264.7 cells

transfected with constitutively active mutant NFATc1 (CA–NFATc1) or control

plasmid for 3 days. RT–PCR (top) and Real–time PCR (bottom). (g) CHIP

assay examining recruitment of NFATc1 to the Lgr4 promoter region. The

putative binding site search was performed by VISTA Genome Browser

(http://genome.lbl.gov/vista/index.shtml).

Supplementary Figure 5. Design of Lgr4flox allele and the bone mass

analysis. (a) Targeting strategy; blank pentagon indicates the inserted loxp

sites. Genomic structure of the wild type murine Lgr4 gene, the targeting

vector and the targeted alleles are indicated. Exons 14–17 are flanked by LoxP

sequences (blank pentagon); the Neo cassette was flanked by frt sites (black

pentagon). The modified Lgr4 locus after homologous recombination

(Neo+allele), and the deleted Lgr4 gene after Cre–mediated excision of exons

14–17 are shown. (b) Representative micro–CT images (n = 5 images taken in

total, one image from 5 different mice each) (left) and parameters (right) of 3D

microCT reconstruction of trabecular bone in Lgr4fl/fl and Lgr4 CKO (Lgr4fl/fl;

LysM–Cre) mice in 8–week–old mice. Error bars, mean ± s.d; *P < 0.05; **P <

0.01; unpaired two–tailed Student's t–test. n = 5 per group. Scale bar, 500 μm.

Supplementary Figure 6. Lgr4 negatively regulates osteoclast

differentiation and bone resorption. (a) Representative TRAP staining


images (n = 4 images taken in total, one image from one well each with

biological duplicate replicate) of In vitro osteoclast formation assay from WT

and Lgr4–/– mice. BMM cells derived from WT and Lgr4–/– mice were cultured

with indicated concentrations of RANKL in the presence of M–CSF (10 ng/ml),

after which the cells were stained for TRAP expression. (b) Representative

images (n 15 images taken in total, one image from one well each with 5

biological replicate) show knockdown of Lgr4 enhanced osteoclast

differentiation in RAW264.7 cells. (c) Representative images (n 30 images

taken in total, 3 image from 1 well each with 10 biological replicate) show

overexpression of LGR4 suppressed osteoclast differentiation in RAW264.7

cells. (d) Comparison of osteoclast-related marker gene expression in wild

type (WT) and Lgr4–/– osteoclasts by RT–PCR analysis. Osteoclast formation

(Nfatc1, Acp5, Src) and resorption (Calcr, Ctsk) markers were analyzed. Scale

bar, 500 μm.

Supplementary Figure 7. Lgr4 deficiency–induced osteoclastogenesis is

independent of RSPOs and Norrin but requires NF–B activation and

GSK3 driven NFATc1 nuclear translocation. (a) BMMs stimulated with

RANKL (100ng/ml) in the presence of M–CSF (10ng/ml) were cultured with

indicated concentrations of RSPO or Norrin, and TRAP+ cells were quantitated.

Error bars, mean ± s.d; n = 3 per group. (b) Representative TRAP staining

images (n = 6 images taken in total, one image from one well each with


triplicated repeated wells of two biological repeated experiment) of RAW264.7

cells stimulated with RANKL (20 ng/ml) and indicated concentrations of

RSPO–1. Representative images are shown (left) and the osteoclasts were

counted (right). Error bars, mean ± s.d; n = 3 per group. (c) Western blot

analysis of IB and phosphorylation of p65 in WT and Lgr4–/– BMMs. Images

are representative of two experiments with biological replicate. (d)

Overexpression of CA–GNAQ suppressed RANKL–induced NFATc1 nuclear

translocation in RAW264.7 preosteoclast cells. NE, nuclear extract; CE,

cytoplasmic extract. Images are representative of more than 3 experiments

with biological replicate. (e) RGS2 (center) or GNAQ–CT (right)

overexpression rescued the suppression of GSK3– phosphorylation by the

RANKL and LGR4 pathway in HEK 293T cells. Images are representative of

two experiments with biological replicate. (f) The phosphorylation of GSK3–

at Ser9 by RANKL is independent of RANK expression. Images are

representative of two experiments with technical replicate. (g) Representative

TRAP staining images (n = 3 images taken in total with technical triplicate

replicate) of mouse BMMs co–cultured with osteoblasts and LGR4–ECD

protein. Error bars, mean ± s.d; *P < 0.05, unpaired two–tailed Student's t–test.

n = 3 per group. (h) Representative TRAP staining images (n = 3 images taken

in total with technical triplicate replicate) show LGR4–ECD protein suppressed

osteoclast formation in primary cultured human giant cell tumor of bone cells.

Error bars, mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3 per group.


Scale bar, 500 μm.

Supplementary Figure 8. Effect of Soluble LGR4–ECD protein on

RANKL–injection bone resorption mouse model and the Tnfrsf11b

knockout (Opg–/–) osteoporosis mouse model. (a-c) Representative images

(n = 6 images taken in total, one image from 6 different mice each) of TRAP

staining (a), representative images (n = 6 images taken in total, one image

from 6 different mice each) of micro–CT (b) and parameter analysis (c) of

mouse whole calvaria obtained from vehicle control (vehicle) or 1 mg/kg/day

RANKL–injected mice treated with either control protein (Ctrl) or LGR4–ECD

(ECD) protein (1 mg/kg/day) daily for two weeks. Arrow indicates area of bone

erosion. Error bars, mean ± s.d; *P < 0.05, **P < 0.01, ***P < 0.001. n = 6 per

group. Scale bar, 2 mm. (d-g) Therapeutic effects of soluble LGR4–ECD

protein on tibia from the Tnfrsf11b knockout (Opg–/–) osteoporosis mouse

model. Representative Micro–CT images (n = 6 images taken in total, one

image from 6 different mice each) (d) and parameter analysis (e),

representative TRAP staining images (n = 18 images taken in total, 3 images

from 6 different mice each) (f) and histomorphometric analysis (g) of mouse

tibia obtained from Tnfrsf11b knockout (Opg–/– mice treated with either PBS

(Ctrl) or LGR4–ECD (ECD) protein (1 mg/kg/day) daily for two weeks. BV/TV,

percent bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number;

Tb.Sp, trabecular separation. Oc.S/BS, osteoclast surface per bone surface;


N.Oc/B.Pm, Osteoclast Bone Surface Density; ES/BS, eroded surface per

bone surface. Error bars, mean ± s.d. *P < 0.05. ***P < 0.001. n = 11 per group.

Scale bar, 500 μm (d) and 100 μm (f). (h) Schematic diagram of LGR4

negative regulation of osteoclastogenesis. RANKL/RANK signaling induces

Lgr4 gene expression, which, on the one hand, can interact with RANKL and

activate the Gq and GSK3– cascade, which inhibits NFATc1 nuclear

translocation, leading to suppression of osteoclastogenesis. On the other hand,

LGR4 competes with RANK for RANKL binding, which results in attenuation of

the canonical RANK cascade. Collectively, LGR4 is a negative feedback

regulator of the RANKL and RANK pathway, suppressing osteoclast

differentiation and bone resorption.

Supplementary Table 1. (Relevant to Figure 3g and 3h) Histomorphometric

parameters of osteoclast in femur bone and calvaria bone. Oc.S/BS, osteoclast

surface per bone surface; N.Oc/B.Pm, Osteoclast Bone Surface Density;

Oc.Size, osteoclast size; ES/BS, eroded surface per bone surface. Error bars,

mean ± s.d; *P < 0.05, **P < 0.01, ***P < 0.001. n = 3 per group.

Supplementary Table 2. Information on PCR Primers.


2016-03-04 supplemental figure.ppt [兼容模式]n-terminal rankl rankl c-terminal n-terminal...

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