Supplementary Information for

Administration of BMP2/7 in utero partially reverses Rubinstein Taybi

Syndrome-like skeletal defects induced by Pdk1 or Cbp mutations in

mice

Jae-Hyuck Shim1,8*, Matthew B. Greenblatt1,2*, Anju Singh1, Nicholas Brady1, Dorothy

Hu1, Rebecca Drapp1, Wataru Ogawa3, Masato Kasuga4, Tetsuo Noda5, Sang-Hwa

Yang6, Sang-Kyou Lee6, Vivienne I. Rebel7, and Laurie H Glimcher1,2,8

1 Department of Immunology and Infectious Diseases, Harvard School of Public Health, and 2 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA 3 Department of Internal Medicine, Division of Diabetes and Endocrinology, Kobe University School of Medicine, Kobe 650-0017, Japan 4 Research Institute, National Center for Global Health and Medicine, Shinjuku-ku, Tokyo, 162-8655, Japan 5 Department of Cell Biology, Cancer Institute, Tokyo 135-8550, Japan 6 Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, NCRIC for Inflammatory Response Modulation, Seoul 120-749, Korea 7 Department of Cellular and Structural Biology, Greehey Children's Cancer Research Institute, UT Health Science Center, San Antonio, TX 78229, USA 8 Correspondence: lglimche@hsph.harvard.edu and jshim@hsph.harvard.edu

* These authors contributed equally to this work

This file contains Supplementary Table S1 and Supplementary Figures S1-S11.

Supplementary Table S1. Genotype of offspring derived from breeding of Pdk1fl/+ osx and Pdk1fl/fl parents Total Flox/Flox Flox/+ Flox/+;Osx Flox/Flox;Osx

P20 and older 134 39 51 42 2 P4-5 29 8 14 7 0 P2-3 76 20 23 14 12 + 7 (dead) P1 67 20 15 14 18

E18.5 47 9 15 4 19 E16.5 32 6 8 10 8

Legends for Supplementary Figures:

Figure S1. Expression of PDK1 in the calvaria.

Immunohistochemistry for PDK1 showing expression in the sagittal suture (A), nasal

bone (B), and zygomatic arch (D) of P2 Pdk1fl/fl and Pdk1osx mice as diagramed. The

osteogenic front (OF) and sutural mesenchyme (SM) are labeled.

Figure S2. Characterization of Pdk1osx mice and embryos.

(A) Pictures of empty stomach in P2 Pdk1fl/fl and Pdk1osx neonates. (B, C) Alizarin

Red/Alcian Blue-stained skeletal preps demonstrating calvarial hypomineralization and

clavicular hypoplasia in E18.5 and E16.5 Pdk1fl/fl and Pdk1osx embryos. Arrows indicate

the zygomatic and nasal bone. (D) Hematoxlin and eosin stained longitudinal section of

the femur in E18.5 and E16.5 Pdk1fl/fl and Pdk1osx embryos. Original magnification 40x.

(E) Alizarin Red/Alcian Blue-stained skeletal preps demonstrating abnormal

skeletogenesis in E18.5 Pdk1fl/fl and Pdk1dm1 embryos. Scale bars indicate 1mm. (F)

Representative FACS profiles on pre-gated live CD45- Ter119- lineage cells harvested

from long bones of E17.5 Pdk1fl/fl and Pdk1osx embryos.

Figure S3. Further characterization of Pdk1dm1 mice .

(A) Alizarin Red/Alcian Blue-stained skeletal preps demonstrating abnormal

skeletogenesis in P0 Pdk1fl/fl and Pdk1dm1 embryos. Scale bars indicate 2mm. (B) Safarin

O-stained sections of tibia from E18.5 Pdk1dm1 embryos. Additionally shown are areas of

delayed cartilage ossifcation with persistent cartilage (top right) and an area of densely-

packed osteoblast-lineage cells embedded in an osteoid matrix (bottom right). Original

magnification of all images 400X.

Figure S4. Characterization of Osx-cre mice.

(A) Alizarin Red/Alcian Blue-stained skeletal preps of calvaria, clavicles, and hind

limbs in P2 Osx-cre neonates. Arrows indicate the zygomatic and nasal bones. (B, C)

Ossification of calvaria and femurs of 4 week old Osx-cre mice was analyzed by µCT.

Displayed are three-dimensional reconstructions of calvaria (C), trabecular bone and

midshaft cortical bone (B, left). Values displayed are bone volume fraction (BV/TV),

trabecular number (Tb.N), trabecular thickness (Tb.Th), and cortical thickness (C. Th).

Values are mean + SD (B, right). (D) Measurement of nonfasting serum glucose (left)

and serum insulin (right) in P2 Pdk1fl/fl and Pdk1osx mice. “*” indicates a significant

difference by the Student’s t-test, p < 0.05.

Figure S5. Impaired osteoblast differentiation in vivo and in vitro cultured CalvObs.

(A) Sections from the nasal bone (upper left), zygoma (right), and calvarium (bottom left)

of P2 Pdk1fl/fl and Pdk1osx neonates were by in situ hybridization. (B) Pdk1fl/fl CalvOb

were transduced with control (con) or Cre-expressing (∆PDK1) lentivirus. RNA

transcript (upper) and protein (day 7, lower) levels of Runx2 were analyzed by

quantitative PCR and immunoblotting with anti-RUNX2 antibody. (C, D) Pdk1fl/fl

CalvOb were transduced with control or Cre-expressing lentivirus and cultured for 7

days. RNA transcript and protein levels of Pdk1 were analyzed by quantitative PCR and

immunoblotting (C). Cell viability was measured with Alamar Blue (upper).

Alternatively, Pdk1fl/fl CalvOb infected by control or Cre-expressing lentivirus were

cultured for 6 days and ALP activity was analyzed (lower) (D). (E, F) Pdk1fl/fl CalvOb

were infected with either control or Cre-expressing lentivirus together with WT or

kinase-dead PDK1 (KD) expressing lentivirus and for ALP activity (E). Alternatively,

RNA transcript levels of the indicated genes were analyzed by quantitative PCR (F).

Figure S6. PI3K/PDK1 inhibitor treatment of CalvOb and hMSCs.

(A) Primary WT CalvObs were cultured in the presence of DMSO, PI3K inhibitor

(LY294002, Calbiochem), PDK1 inhibitor (B-3012, Echelon Biosciences) or AKT

inhibitor (IV, Calbiochem). (B, C) hMSCs were treated with PDK1 or PI3K inhibitors for

6 days while placed under osteoblast differentiation conditions. They were then assayed

for ALP activity (B) or for expression of osteoblast marker genes (C).

Figure S7. Various signaling pathways in PDK1-deficient osteoblasts.

(A) Pdk1fl/fl CalvOb were infected with control (con) or Cre-expressing (∆PDK1)

lentivirus, cultured for 6 days under differentiation conditions in the absence or presence

of IGF-1 (25ng/ml), and analyzed by Von Kossa staining for mineralization. (B) FGF

signaling transduction in PDK1-deficient CalvObs. Pdk1fl/fl CalvOb were infected with

control or Cre-expressing lentivirus and cultured under differentiation conditions. RNA

transcript levels of Sprouty1 (Spry1) and Sprouty2 (Spry2) were analyzed by quantitative

PCR. (C) BMP signaling transduction in PDK1-deficient CalvObs. The cells were treated

with or without BMP2/7 (100ng/ml) for 6 hours and RNA transcript levels of the

indicated genes were analyzed by quantitative PCR. (D) TGFβ signaling transduction in

PDK1-deficient CalvObs. The cells were transfected with 3TP-lux and Renilla luciferase

gene, and luciferase activity was analyzed 6 days after culture in differentiation medium.

Results were normalized to a Renilla control. (E) PDK1-deficient CalvObs were serum

starved for 12 hours prior to stimulation with FGF2 (10ng/ml), BMP2/7 (100ng/ml), or

TGFβ (2ng/ml) at different timepoints and immunoblotted with the indicated antibodies.

Protein amount of HSP90 was used as a loading control. (F) PDK1-deficient CalvObs

were stimulated with IGF-1 (25ng/ml) and insulin (10nM) at different timepoints and

immunoblotted with the indicated antibodies. Protein amount of HSP90 was used as a

loading control. These experiments were performed together with Figures 2E and F. (G)

C3H10T1/2 osteoblasts were transduced with the Top flash lucfierase reporter

concurrently with the construct encoding xWNT/Fz. These cells were concurrently

treated with the indicated inhibitors and activity reported 36 hours later. (H) CalvOb were

prepared as in (A) and then blotted for β-Catenin (bottom panel). Alternatively, the

activity of β-Catenin was examined with the Top flash luciferase reporter (top panel).

Figure S8. Impaired AKT activation in Pdk1osx mice.

(A) WT CalvObs were treated with the indicated inhibitors for 1hour prior to stimulation

with IGF-1 (25ng/ml) for 30 minutes and immunoblotted with antibodies specific to

phospho-AKT (T308) and AKT substrates. Protein amount of HSP90 was used as a

loading control. (B, C, D) IHC showing phosphorylation levels of AKT (T308), GSK3β

(S9), and S6 (S235/236) in a coronal section of the sagittal suture (B), nasal bone (C),

and zygomatic arch (D) of P2 Pdk1fl/fl and Pdk1osx neonates. Original magnification 100X.

(E) IHC showing phosphorylation levels of CREB (S133) in a coronal section of the

nasal bone (left) and zygomatic arch (right) of P2 Pdk1fl/fl and Pdk1osx neonates. Original

magnification 100X.

Figure S9. Characterization of CREB knockout mice.

(A) Transcript levels of Creb and Cbp in PDK1-deficient osteoblasts. Pdk1fl/fl CalvOb

were infected with control (con) or Cre-expressing (∆PDK1) lentivirus and cultured

under differentiation conditions. RNA transcript levels of the indicated genes were

analyzed by quantitative PCR. (B) Alizarin Red/Alcian Blue-stained skeletal preps of

E18.5 Creb+/+ and Creb-/- embryos. (C) µCT analysis of calvaria (upper left; right) and

Alizarin Red/Alcian Blue-stained skeletal preps of the clavicles (lower left) from 2 week

old Pdk1fl/+, Pdk1fl/+osx, Creb+/-, and Pdk1fl/+osx; Creb+/- mice. (D) CREB

phosphorylation by various stimuli in PDK1-deficient CalvObs. The cells were

stimulated with FGF-2 (top), isoproterenol (middle), or forskolin (bottom) at different

timepoints and immunoblotted with anti-phospho-CREB (S133) antibody. Protein

amount of HSP90 was used as a loading control. (E) CalvOb were prepared as in Figure

S9A and their size determined by the forward-scatter on a flow cytometer.

Figure S10. PDK1 and CREB regulate BMP2 expression.

(A) hMSCs (top panel) or CalvOb (bottom panel) were cultured under osteoblast

differentiating conditions and treated with the indicated inhibitors for 24 hours and the

transcript levels of BMP2 measured by quantitative PCR. (B) Transcript levels of Bmp2

in PDK1-deficient osteoblasts. Pdk1fl/fl CalvOb were infected with control (con) or Cre-

expressing (∆PDK1) lentivirus and cultured under differentiation conditions. RNA

transcript levels of the indicated genes were analyzed by quantitative PCR. (C)

C3H10T1/2 cells were transfected with the BMP2-Luc (-150/+165) and Renilla luciferase

genes along with a different concentration of VP16/CREB (top panel) or with the

combination of Flag-CREB (wt) and CBP (bottom panel). Results are expressed as

relative luciferase activity normalized to Renilla control. (D) C3H10T1/2 cells were

transfected with the BMP2-Luc (-2712/+165 or -150/+165) and Renilla luciferase genes

along with vector, Flag-CREB (wt), or VP16/CREB. Results are expressed as relative

luciferase activity normalized to Renilla control.

Figure S11. Quantification of Western blotting results.

The relative band intensity from the indicated Western blots was measured. The second

set of values reflects the results of an independent replicate experiment.

Pdk1fl/fl Pdk1osx

Pdk1fl/fl

Pdk1osx

SMOF

OF

SM

Pdk1fl/fl Pdk1osx

A

B

C

SM

OF

SMOF

OcnPdk1fl/fl

Pdk1osx

D

Figure S1. Expression of PDK1 in the calvaria.

Immunohistochemistry for PDK1 showing expression in the sagittal suture (A), nasal bone (B), and

zygomatic arch (D) of P2 Pdk1fl/fl and Pdk1osx mice as diagramed. The osteogenic front (OF) and sutural

mesenchyme (SM) are labeled.

Pdk1fl/fl Pdk1osx

A Pdk1fl/fl

Pdk1osx

E16.5

Pdk1fl/fl Pdk1osx Pdk1fl/fl

Pdk1osx

E18.5

Pdk1fl/fl Pdk1osx

Pdk1fl/fl Pdk1osx

E18.5

E16.5

B C

D

E F276%

223%

362%

381%

CD105

Thy1

.2

Pdk1osx

Pdk1fl/fl

Pdk1osx

Pdk1fl/fl

Thy1.2+ Thy1.2-

CD105+

Cell

num

ber

0

0.5

1

1.5

2

2.5

3

Figure S2. Characterization of Pdk1osx mice and embryos.

(A) Pictures of empty stomach in P2 Pdk1fl/fl and Pdk1osx neonates. (B, C) Alizarin Red/Alcian Blue-stained skeletal preps demonstrating calvarial

hypomineralization and clavicular hypoplasia in E18.5 and E16.5 Pdk1fl/fl and Pdk1osx embryos. Arrows indicate the zygomatic and nasal bone. (D) Hematoxlin

and eosin stained longitudinal section of the femur in E18.5 and E16.5 Pdk1fl/fl and Pdk1osx embryos. Original magnification 40x. (E) Alizarin Red/Alcian Blue-

stained skeletal preps demonstrating abnormal skeletogenesis in E18.5 Pdk1fl/fl and Pdk1dm1 embryos. Scale bars indicate 1mm. (F) Representative FACS

profiles on pre-gated live CD45- Ter119- lineage cells harvested from long bones of E17.5 Pdk1fl/fl and Pdk1osx embryos.

Pdk1fl/fl Pdk1dm1

Pdk1fl/fl

Pdk1dm1

Pdk1fl/fl Pdk1dm1

Pdk1fl/fl Pdk1dm1

A

Pdk1fl/fl Pdk1dm1

B

Figure S3.Further characterization of Pdk1dm1 mice .

(A). Alizarin Red/Alcian Blue-stained skeletal preps demonstrating abnormal skeletogenesis in P0 Pdk1fl/fl and

Pdk1dm1 embryos. Scale bars indicate 2mm. (B) Safarin O-stained sections of tibia from E18.5 Pdk1dm1 embryos.

Additionally shown are areas of delayed cartilage ossifcation with persistent cartilage (top right) and an area of densely-

packed osteoblast-lineage cells embedded in an osteoid matrix (bottom right). Original magnification of all images 400X.

Pdk1fl/fl Pdk1dm1

Pdk1dm1

A

B

+/+ Osx

+/+

Osx

+/+ OsxC

+/+ Osx Pdk1fl/osx

Tb

.N (

mm

-1)

BV

/TV

(%

)

+/+ Osx Pdk1fl/osx

Tb

.Th

(m

m)

+/+ Osx Pdk1fl/osx +/+ Osx Pdk1fl/osx

C.T

h (

mm

)

+/+ Osx

+/+ Osx

Figure S4. Characterization of Osx-cre mice.

(A) Alizarin Red/Alcian Blue-stained skeletal preps of calvaria, clavicles, and hind limbs in P2 Osx-cre

neonates. Arrows indicate the zygomatic and nasal bones. (B, C) Ossification of calvaria and femurs of 4

week old Osx-cre mice was analyzed by µCT. Displayed are three-dimensional reconstructions of calvaria

(C), trabecular bone and midshaft cortical bone (B, left). Values displayed are bone volume fraction

(BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and cortical thickness (C. Th). Values are

mean + SD (B, right). (D) Measurement of nonfasting serum glucose (left) and serum insulin (right) in P2

Pdk1fl/fl and Pdk1osx mice. “*” indicates a significant difference by the Student’s t-test, p < 0.05.

Seru

m insulin

Pdk1fl/fl Pdk1osx

*

0

20

40

60

80

100

120

140

160

Seru

m g

lucose

Pdk1fl/fl Pdk1osx

D

Pdk1fl/fl Pdk1osx

AC

ell

via

bili

ty

con ∆PDK10

100

200

300

400

500

600

700

800

900

B C

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

con ∆PDK1

Pdk1 tra

nscrip

t/H

PR

T

PDK1

GAPDH

con ∆PDK1

IB

E

Bsp

Ocn

Col1

0

0.00005

0.0001

0.00015

0.0002

0.00025

0

2

4

6

8

10

12

14

16

18

20

0

0.001

0.002

0.003

0.004

0.005

0.006

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

Osx

Tra

nscrip

t le

ve

l/H

PR

T

con WT KD

FPdk1fl/fl

Pdk1osx

Pdk1osx

Pdk1fl/fl

Opn

Ocn

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

ALP

activity/a

lam

ar

blu

e

con ∆PDK1

D

con ∆PDK1 con ∆PDK1

IB: RUNX2 PDK1

con

∆PDK1

Runx2 t

ranscrip

t/H

PR

T

0

0.2

0.4

0.6

0.8

1

1.2

1.4

d7 d14

con WT KD

ALP

activity/a

lam

ar

blu

e

WT

∆PDK1

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

con

con

WT

KD

∆PDK1

Figure S5. Impaired osteoblast differentiation in vivo and in vitro cultured CalvObs.

(A) Sections from the nasal bone (upper left), zygoma (right), and calvarium (bottom left) of P2 Pdk1fl/fl and Pdk1osx neonates were by in situ hybridization. (B) Pdk1fl/fl CalvOb were transduced with control (con) or Cre-

expressing (PDK1) lentivirus. RNA transcript (upper) and protein (day 7, lower) levels of Runx2 were analyzed by quantitative PCR and immunoblotting with anti-RUNX2 antibody. (C, D) Pdk1fl/fl CalvOb were

transduced with control or Cre-expressing lentivirus and cultured for 7 days. RNA transcript and protein levels of Pdk1 were analyzed by quantitative PCR and immunoblotting (C). Cell viability was measured with

Alamar Blue (upper). Alternatively, Pdk1fl/flCalvOb infected by control or Cre-expressing lentivirus were cultured for 6 days and ALP activity was analyzed (lower) (D). (E, F) Pdk1fl/flCalvOb were infected with either

control or Cre-expressing lentivirus together with WT or kinase-dead PDK1 (KD) expressing lentivirus and for ALP activity (E). Alternatively, RNA transcript levels of the indicated genes were analyzed by quantitative

PCR (F).

SM OF

SMOF

Ocn

Pdk1fl/fl

Pdk1osx

Opn

Ocn

0

0.0005

0.001

0.0015

0.002

0.0025

ALP

activity/a

lam

ar

blu

e

Inh: -

PI3K-inh PDK1-inh

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

0.0018

ALP

activity/a

lam

ar

blu

e

DMSO AKT-inh

PI3K-inh

PDK1-inh

DMSO

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

0.0018

PI3K-inh: - +

ALP activity/alamar blue

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

0.0018

PDK1-inh: - +

Tra

nscript le

vel/H

PR

T

A

B C

0

0.5

1

1.5

2

2.5

3

3.5

Bsp

0

5

10

15

20

25

Col1

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

Ocn

0

0.01

0.02

0.03

0.04

0.05

0.06

Osx

Figure S6. PI3K/PDK1 inhibitor treatment of CalvOb and hMSCs.

(A) Primary WT CalvObs were cultured in the presence of DMSO, PI3K inhibitor (LY294002, Calbiochem), PDK1 inhibitor (B-3012, Echelon Biosciences) or

AKT inhibitor (IV, Calbiochem). (B, C) hMSCs were treated with PDK1 or PI3K inhibitors for 6 days while placed under osteoblast differentiation conditions.

They were then assayed for ALP activity (B) or for expression of osteoblast marker genes (C).

A BC

0

2000

4000

6000

8000

10000

12000

3T

P-L

uc

con ∆PDK1

P-SMAD2

HSP90

TGF: 0 15 30 60 0 15 30 60 (min)

con ∆PDK1

IB

E

P-GSK3

GAPDH

P-S6

IB

IGF-1: 0 15 30 60 0 15 30 60 (min)

con ∆PDK1

HSP90

IB

P-S6

P-GSK3

Insulin: 0 15 30 60 0 15 30 60 (min)

con ∆PDK1

None IGF1

Von Kossa

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0

0.05

0.1

0.15

0.2

0.25

0.3

Tra

nscript le

vel/H

PR

T

Sprouty1

Sprouty2

con ∆PDK1

HSP90

FGF2: 0 5 30 60 0 15 30 60 (min)

con ∆PDK1

IB

P-ERK1/2

0

0.5

1

1.5

2

2.5

None

BMP2/7

Tra

nscript le

vel/H

PR

T

Noggin

0

0.5

1

1.5

2

2.5

Id1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Osx

con ∆PDK1

BMP2/7: 0 15 30 60 0 15 30 60 (min)

HSP90

IB

P-SMAD

1/5/8

con ∆PDK1

con

∆PDK1

D

F

Figure S7. Various signaling pathways in PDK1-deficient osteoblasts.

(A) Pdk1fl/fl CalvOb were infected with control (con) or Cre-expressing (PDK1) lentivirus, cultured for 6 days under differentiation conditions in the absence or presence of IGF-1 (25ng/ml), and analyzed by Von Kossa staining for

mineralization. (B) FGF signaling transduction in PDK1-deficient CalvObs. Pdk1fl/fl CalvOb were infected with control or Cre-expressing lentivirus and cultured under differentiation conditions. RNA transcript levels of Sprouty1

(Spry1) and Sprouty2 (Spry2) were analyzed by quantitative PCR. (C) BMP signaling transduction in PDK1-deficient CalvObs. The cells were treated with or without BMP2/7 (100ng/ml) for 6 hours and RNA transcript levels of the

indicated genes were analyzed by quantitative PCR. (D) TGF signaling transduction in PDK1-deficient CalvObs. The cells were transfected with 3TP-lux and Renilla luciferase gene, and luciferase activity was analyzed 6 days

after culture in differentiation medium. Results were normalized to a Renilla control. (E) PDK1-deficient CalvObs were serum starved for 12 hours prior to stimulation with FGF2 (10ng/ml), BMP2/7 (100ng/ml), or TGF (2ng/ml) at

different timepoints and immunoblotted with the indicated antibodies. Protein amount of HSP90 was used as a loading control. (F) PDK1-deficient CalvObs were stimulated with IGF-1 (25ng/ml) and insulin (10nM) at different

timepoints and immunoblotted with the indicated antibodies. Protein amount of HSP90 was used as a loading control. These experiments were performed together with Figures 2E and F. (G) C3H10T1/2 osteoblasts were

transduced with the Top flash lucfierase reporter concurrently with the construct encoding xWNT/Fz. These cells were concurrently treated with the indicated inhibitors and activity reported 36 hours later. (H) CalvOb were

prepared as in (A) and then blotted for β-Catenin (bottom panel). Alternatively, the activity of β-Catenin was examined with the Top flash luciferase reporter (top panel).

G

GAPDH

-Catenin

IBcon ∆PDK1

0

5000

10000

15000

20000

25000

30000

35000

con ∆PDK1

Top fla

sh L

uc

H

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

To

p-f

lash

Lu

c

xWNT/Fz: - + + + + + + +

PI3K inh PDK1 inh

AC

P-AKT

(T308)

SM

P-S6

(S235/236)

P-GSK3

(S9)

P-AKT

(T308)

Pdk1fl/fl Pdk1osx

D

Pdk1fl/fl

Pdk1osx

Pdk1fl/fl Pdk1osx

Pdk1fl/fl Pdk1osx Pdk1fl/fl Pdk1osx Pdk1fl/fl Pdk1osx

P-AKT (T308) P-S6 (S235/236)E

P-CREB (S133)P-CREB (S133)

P-GSK3

(S9)

P-S6

(S235/236)

P-GSK3 (S9)

HSP90

P-AKT (T308)

Inhibitor: - -

None IGF-1

P-AKT

substrates

250kD

150kD

100kD

75kD

50kD

35kD

B

SM

SM

SM

SM

SM

OF

OF

OF

OF

OF

OF

Figure S8. Impaired AKT activation in Pdk1osx mice.

(A) WT CalvObs were treated with the indicated inhibitors for 1hour prior to stimulation with IGF-1

(25ng/ml) for 30 minutes and immunoblotted with antibodies specific to phospho-AKT (T308) and AKT

substrates. Protein amount of HSP90 was used as a loading control. (B, C, D) IHC showing

phosphorylation levels of AKT (T308), GSK3 (S9), and S6 (S235/236) in a coronal section of the

sagittal suture (B), nasal bone (C), and zygomatic arch (D) of P2 Pdk1fl/fl and Pdk1osx neonates. Original

magnification 100X. (E) IHC showing phosphorylation levels of CREB (S133) in a coronal section of

the nasal bone (left) and zygomatic arch (right) of P2 Pdk1fl/fl and Pdk1osx neonates. Original

magnification 100X.

Creb: +/+ -/- +/+ -/-

Creb+/+

Creb-/-

Pdk1fl/+osx

Creb+/-

Pdk1fl/+ Pdk1fl/+osx Pdk1fl/+ Pdk1fl/+osx

Creb+/+ Creb+/+ Creb+/- Creb+/-

Pdk1fl/+

Creb+/+

Pdk1fl/+osx

Creb+/+

Pdk1fl/+

Creb+/-

0

0.005

0.01

0.015

0.02

0.025T

ranscript le

vel/H

PR

T Creb

con ∆PDK1

0

0.5

1

1.5

2

2.5

3

Cbp

B

IB

IB

FGF2: 0 15 30 60 0 15 30 60 (min)

HSP90

P-CREB

con ∆PDK1

GAPDH

P-CREB

GAPDH

P-CREB

ISO: 0 20 40 0 20 40 (min)

FSK: 0 20 40 0 20 40 (min)

IB

con ∆PDK1

con ∆PDK1

A

C

D

Figure S9. Characterization of CREB knockout mice.

(A) Transcript levels of Creb and Cbp in PDK1-deficient osteoblasts. Pdk1fl/fl CalvOb were infected with control (con) or Cre-expressing (PDK1) lentivirus and cultured under differentiation conditions. RNA transcript

levels of the indicated genes were analyzed by quantitative PCR. (B) Alizarin Red/Alcian Blue-stained skeletal preps of E18.5 Creb+/+ and Creb-/- embryos. (C) CT analysis of calvaria (upper left; right) and Alizarin

Red/Alcian Blue-stained skeletal preps of the clavicles (lower left) from 2 week old Pdk1fl/+, Pdk1fl/+osx, Creb+/-, and Pdk1fl/+osx; Creb+/- mice. (D) CREB phosphorylation by various stimuli in PDK1-deficient CalvObs.

The cells were stimulated with FGF-2 (top), isoproterenol (middle), or forskolin (bottom) at different timepoints and immunoblotted with anti-phospho-CREB (S133) antibody. Protein amount of HSP90 was used as a

loading control. (E) CalvOb were prepared as in Figure S9A and their size determined by the forward-scatter on a flow cytometer.

E

0 200 400 600 800 1000FSC-H: FSC-Height

0

20

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% o

f M

ax

con∆PDK1

CBP

BM

P2

-lu

c

0

100000

200000

300000

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-2712/+165 -150/+165

A

B

0

5000

10000

15000

20000

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30000

BM

P2

-luc

(-1

50

/+16

5)

CREB (wt): - + - +

C

D

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

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0.009

0.01

BM

P2 tra

nscript/H

PR

T

con ∆PDK1

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0.005

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0.03

0.035

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VP16/CREB: -

BM

P2

-luc

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5)

Inh: -PI3K-inh PDK1-inh

hMSC

0

0.001

0.002

0.003

0.004

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0.007

CalvOb

BM

P2 tra

nscript/H

PR

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Inh: -PI3K-inh PDK1-inh

Pdk1fl/fl CalvOb

BM

P2 tra

nscript/H

PR

T

Figure S10. PDK1 and CREB regulate BMP2 expression.

(A) hMSCs (top panel) or CalvOb (bottom panel) were cultured under osteoblast differentiating conditions and treated with the indicated

inhibitors for 24 hours and the transcript levels of BMP2 measured by quantitative PCR. (B) Transcript levels of Bmp2 in PDK1-deficient

osteoblasts. Pdk1fl/fl CalvOb were infected with control (con) or Cre-expressing (PDK1) lentivirus and cultured under differentiation conditions.

RNA transcript levels of the indicated genes were analyzed by quantitative PCR. (C) C3H10T1/2 cells were transfected with the BMP2-Luc (-

150/+165) and Renilla luciferase genes along with a different concentration of VP16/CREB (top panel) or with the combination of Flag-CREB

(wt) and CBP (bottom panel). Results are expressed as relative luciferase activity normalized to Renilla control. (D) C3H10T1/2 cells were

transfected with the BMP2-Luc (-2712/+165 or -150/+165) and Renilla luciferase genes along with vector, Flag-CREB (wt), or VP16/CREB.

Results are expressed as relative luciferase activity normalized to Renilla control.

P-AKT

(T308)

0.89

0.89

0.92

1

1

0.33

0.97

0.37

0.27

0

0.15

0

0.21

0

0.3

0

P-AKt

(S473)

0.22

0.14

0.95

0.9

0.60

0.88

0.64

0.44

0.33

0.53

0.97

1

0.92

0.97

1

0.95

P-p380.4

0.18

0.19

0.7

0.15

0.7

0.23

0.72

0.89

0.75

0.96

0.84

1

0.92

0.83

1

GAPDH0.94

0.89

0.96

0.99

0.95

0.86

0.94

0.9

1

0.98

0.96

1

0.92

1

1

0.98

Figure. 2E

P-AKT

(T308)

1

1

0.11

0.11

P-AKT

(S473)

1

1

1.32

1.8

GAPDH1

1

0.98

1

P-GSK31

1

0.28

0.31

P-S61

1

0

0

Figure. 3A

P-CREB

(S133)

1

1

0.43

0.46

HSP901

0.86

0.98

1

Figure. 3B

P-CREB

(S133)

0.19

0

1

1

0.47

0.77

0.31

0.77

0.09

0.11

0.16

0.15

0.14

0.11

0

0

HSP901

0.87

0.99

0.92

0.96

0.93

0.95

0.91

0.94

0.94

0.93

0.93

0.94

0.93

0.96

1

Figure. 3D

Oligo

pulldown

1

1

0.42

0.34

2.13

2.99

0.64

1.14

Input1

1

1

1.03

0.99

1.01

1.05

1.01

Figure. 5B

IP:HA

IB: Myc

0

0

0.53

0.49

1

1

IB: Myc0.97

1

1

0.96

1

0.97

Figure. 5C

IP:IgG, CBP

IB: Runx2

0.11

0.18

1

1

0.46

0.57

IB: Runx20.81

0.84

1

1

0.99

1.03

Figure. 5D

Oligo

pulldown

1

1

0.36

0.41

Input1

0.98

1

1

Figure. 5I

P-SMAD

1/5/8

1

1

0.49

0.32

GAPDH1

1

0.97

0.96

Figure. 5F

Figure S11. Quantification of Western blotting results.

The relative band intensity from the indicated Western blots was measured. The second set of values reflects the results of an independent replicate experiment.