a

Supplementary Figure 1

b

c

CN MN Ctrl0

5

10

iNAD

+ (n

M)

G-CSF + + +

*

0

350

700

1050

1400

C/EB

Pβ/β

-act

in

mRN

A ra

tio (A

U)

G-CSF - + + + - + CNCN IN Ctrl MN Ctrl

*

0

0.25

0.501

3

NAM

PT p

rote

in

(ng

ml-1

pla

sma)

G-CSF - + + + - +CNCN IN Ctrl MN Ctrl

2

**

*

0153045

75 bone marrowpromyelocytes

G-CSF - + - +CNCN Ctrl Ctrl

NAM

PT/ß

-act

in

mRN

A ra

tio (A

U)

**

*

60

*

d

NAMPT is essential for the G-CSF induced myeloid differentiation via anovel NAD+- /SIRT1- dependent pathway

J Skokowa, D Lan, BK Thakur, F Wang, K Gupta, G Cario, A Müller Brechlin, A Schambach,L Hinrichsen, G Meyer, M Gaestel, M Stanulla, Q Tong, K Welte

Supplementary Information

Supplementary Figures

1

Nature Medicine: doi:10.1038/nm.1913

2

Nature Medicine: doi:10.1038/nm.1913

CNCtrl

a

Supplementary Figure 2

c

d

0

2

4

6

NAM

PT p

rote

in(n

g m

l-1 cu

lture

med

ium

)

2 h 4 h

0

10203040

2 h 4 h

NAM

PT/β

-act

in m

RNA

ratio

(AU)

G-CSF - + - + - + -+

G-CSF - + - + - + -+

0

3.5

7.02 h 4 h

iNAD

+ (n

M)

G-CSF - + - + - + -+

0

1

345

CD33

+ c

ells

eNAD

+ (n

M)

02468

10

PMN

seN

AD+, (

nM)**

**

*

G-CSF - + - + G-CSF - + - +

b CNCtrl

CNCtrl

CNCtrl

**

*

****

*

*

**

**

2

3

Nature Medicine: doi:10.1038/nm.1913

4

Nature Medicine: doi:10.1038/nm.1913

Supplementary Figure 3

d

Ctrl lv NAMPT

NAMPT lvGCSF

c

NAMPT

CtrlCtrl-lv

NAMPT-lv

a

0 4 8 120

20

40

60

Perc

enta

ge o

f CD

16+ c

ells

Time (d)

NAMPT lvCtrl lv

30

0

15

CE

BPA

/ß-a

ctin

mRN

A ra

tio (A

U) *

0

100

200 *

0

10

20 *

Ctrl lv NAMPT lvIsotype9% 52% 45% 67%

GFP

CD11

b 0.3%

NAMPTCtrl

iNAD

+ (n

M)

0

30

60 *

S

IRT1

/ß-a

ctin

mRN

A ra

tio (A

U)

C

EBPB

/ß-a

ctin

mRN

A ra

tio (A

U)

b

5

Nature Medicine: doi:10.1038/nm.1913

6

Nature Medicine: doi:10.1038/nm.1913

Supplementary Figure 4

c

aNAMPTCtrl

G-CSFNAMPT + G-CSF

0 7 10 140

5

10

15

Time (d)

Ctrl lv + NAMPT

Ctrl + G-CSFCtrl lv + G-CSF

NAMPT lv + G-CSF

0 4 8 120

5

10

Time (d)

CD16

+ c

ells

, x 1

04CD

16+ c

ells

, x 1

04

- NAMPT + NAMPT

b

10 μm 10 μm

7

Nature Medicine: doi:10.1038/nm.1913

8

Nature Medicine: doi:10.1038/nm.1913

a b

0 2 4 60

15

30

45

60

Perc

enta

ge o

f CD

16+ c

ells

Time (d)

NAMPTATRAG-CSFCtrl

Supplementary Figure 5

- NAMPT

+ NAMPTNAM

PT lv

mock

SIRT1

C/EBPβ

NAMPT

β-actin

d

CEBPα

NAMPT

β-actin

NAMPT

lv lv

mock

c

SIR

T1/ß

-act

in m

RNA

ratio

(AU)

0

4

8

12 **

0

100

200

300

*

0

30

60*

+ NAMPTCtrl

+ ATRA+ NAMPT lv

* *

**

CE

BPA/

ß-ac

tin m

RNA

ratio

(AU)

CE

BPB/

ß-ac

tin m

RNA

ratio

(AU)

10 μm

10 μm

110 kDa

55 kDa

37.5 kDa

45 kDa

55 kDa

45 kDa

55 kDa

40 kDa

9

Nature Medicine: doi:10.1038/nm.1913

10

Nature Medicine: doi:10.1038/nm.1913

Supplementary Figure 6

c

B

CtrlG-

CSFR

shR

NA

RFP

CD15isotype

0% 4.1%

0.5% 4.1%

0.2% 35%

+ NAMPT

+ GM-CSF

a

048

12205080

120

* * *

FK866 - + - + - +

ControlG-CSFNAMPT

G-C

SF(n

g m

l-1 m

ediu

m)

0.4

0.8

1.2

*

FK866 - +0

ControlNAMPT

CSF3

/ß-a

ctin

mRN

A ra

tio (A

U)

b

FK866 - + - + - +

ControlG-CSFNAMPT

0

3

6

9

**iN

AD+, (

nM)

11

Nature Medicine: doi:10.1038/nm.1913

12

Nature Medicine: doi:10.1038/nm.1913

Supplementary Figure 7

NAMPTcDNA

NAMPT cDNA

+---

+---

+--

-+

-

-

SIRT1 siRNASIRT1

- - - -

NAD+-

-

-

-

- +-

-

-

-

+

- - - - rhNAMPT - -- - - - - -

0

0.1

0.2

0

0.1

0.2

rhNAMPT

CEBPBCEBPA

- - - -- - + -- - - +- - - -

-+-

--+-

++---

---

++--

- + - + +- + - -+ + - -- - + +

+-+

-+-+

--++-

--+

---+

-

CSF3 promoter with two C/EBP binding sitesCSF3 promoter with two mutated C/EBP binding sites

-----

--

-----

--

--+-

+-+-

-+-

+

- - -

-

+

-

-

+

- - - ++ - - -

--+--

+-

--+--

+-

--+--

-+

-

+

-

-

+

+-

-

-

-

+

+

+-

-

-

-

+

+

+-

c

b

RLU

RLU

CSF3 promoter with two C/EBP binding sitesCSF3 promoter with two mutated C/EBP binding sites

* * * * * * **

**

CEBPBCEBPA

G-CSF promoter– 411 bpC/EBP

AGATTCCACAATTTCACAAAACT

G-CSF– 388 bpC/EBP

a

13

Nature Medicine: doi:10.1038/nm.1913

14

Nature Medicine: doi:10.1038/nm.1913

Supplementary Figure 8

0

0.1

0.2

0

0.1

0.2

NAMPT cDNA rhNAMPT - - - -

- - + -- - - +- - - -

-+-

--+-

++---

---

++--

- + - + +- + - -+ + - -- - + +

+-+

-+-+

--++-

--+

---+

-

+---

+---

+--

-+

-

-

SIRT1 siRNASIRT1

- - - -

NAD+

-

-

-

-

- +-

-

-

-

+

- - - - rhNAMPT - -- - - - NAMPT cDNA - -

-----

--

-----

--

--+-

+-+-

-+-

+

- - -

-

+

-

-

+

- - - ++ - - -

--+--

+-

--+--

+-

--

+--

-+

-

+

-

-

+

+-

-

-

-

+

+

+-

-

-

-

+

+

+-

c

b

RLU

RLU

CSF3R promoter with two C/EBP binding sitesCSF3R promoter with two mutated C/EBP binding sites

CSF3R promoter with two C/EBP binding sitesCSF3R promoter with two mutated C/EBP binding sites

* * ** **

*

*

*

CEBPBCEBPA

CEBPBCEBPA

G-CSFR promoter– 1297 bpC/EBP

GGGTTTGGGTAAGACG-CSFR–208 bp

C/EBPGACTTGCATCATTTT

a

15

Nature Medicine: doi:10.1038/nm.1913

16

Nature Medicine: doi:10.1038/nm.1913

Supplementary Figure 9

a+ Vitamin B3Ctrl

0

100

200

300

0

50

100

0

200

400

600 ** *

0

10

20

30

40

0

25

50

75

0

25

50

75

CEBP

A/ß-

actin

mRN

A ra

tio (A

U)

CEBP

A/ß-

actin

mRN

A ra

tio (A

U)

CSF3

R/ß-

actin

mRN

A ra

tio (A

U)

CEBP

B/ß-

actin

mRN

A ra

tio (A

U)

CEBP

B/ß-

actin

mRN

A ra

tio (A

U)

CSF3

R/ß-

actin

mRN

A ra

tio (A

U)

b + Vitamin B3Ctrl

17

Nature Medicine: doi:10.1038/nm.1913

18

Nature Medicine: doi:10.1038/nm.1913

19

Nature Medicine: doi:10.1038/nm.1913

20

Nature Medicine: doi:10.1038/nm.1913

21

Nature Medicine: doi:10.1038/nm.1913

22

Nature Medicine: doi:10.1038/nm.1913