0

500

1000

1500

2000

2500

3000

3500

4000

11:21

:55

12:39

13:53

15:07

16:20

17:34

18:48

20:01

21:15

22:29

23:42 0:5

62:1

03:2

34:3

75:5

17:0

48:1

89:3

210

:4511

:59

Time

Am

bula

tory

Cou

nts

BMP7-adenoControl-adenoDark Cycle

Supplemental Figure S1

B. DIO mice C. DIO mice

0

50

100

150

200

250

Control BMP7

Ser

um A

LT (U

/L)

N.S.

0

20

40

60

80

100

120

Control BMP7

Ser

um I

L6 (p

g/m

L)

N.S.

D. ob/ob mice E. ob/ob mice

Control BMP70

200400600800

100012001400160018002000

Ser

um A

LT (U

/L)

*

0

20

40

60

80

100

120

Control BMP7

Ser

um I

L6 (p

g/m

L)

N.S.

A. Activity

Supplemental Figure S1(A) Activity data from mice treated with control or BMP7-adenovirus, showing no difference in locomotion as measured by ambulatory counts in CLAMS.(B) Liver ALT levels were not increased by BMP7-adenovirus in DIO mice. (N.S. = not significant), indicating mice did not become ill from the treatment.(C) Circulating IL6 levels were not increased by BMP7-adenovirus in DIO mice. (N.S. = not significant), indicating mice did not become ill from the treatment.(D) Liver ALT levels were decreased in BMP7-adenovirus-treated ob/ob mice compared to control LacZ-adenovirus treated ob/ob mice, indicating mice did not become ill from the treatment.(E) Circulating IL6 levels were not increased by BMP7-adenovirus in ob/ob mice, indicating mice did not become ill from the treatment.

Supplemental Figure S2

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Control BMP2Food

con

sum

ed in

4hr

s (g

) afte

r i.c

.v. B

MP

2A. Food Intake after i.c.v. BMP2

0

20

40

60

80

100

120

i.c.v. vehicle

i.c.v. BMP7

i.p. vehicle

i.p. LiCl

Sac

char

in P

refe

renc

e

*

C. Conditioned Taste Aversion

B. Average mouse malaise scores after i.c.v. BMP7 or vehicle

00 Total Score

  No malaiseNo malaisescore greater than

1 should be considered

malaise

Normal: eats dry food, drinks water, evidence of urine and feces.0 – 0.40 0Appetite

Less than 5% weight loss over preoperative weight.0 – 0.40 0Weight

Normal – no ataxia, hunching or piloerection0 – 0.40 0Gait and postures

None.0 – 0.40 0Porphyrin staining

Bright and alert – not burrowing or hiding; moving around cage

normally, responds normally to touch.0 – 0.40 0Attitude

DetailsScore rangeIndividual Score

i.c.v. vehicle (average of 16 mice)

Individual Scorei.c.v. BMP7

(average of 16 mice)Observation

Supplemental Figure S2(A) Mice treated i.c.v. with BMP2 showed no difference in acute food intake versus mice receiving i.c.v. vehicle treatment.(B)Average mouse malaise scores for BMP7-treated mice (i.c.v. administration) vs. vehicle-treated controls, showing no illness in BMP7-treated mice (average of 16 mice tested for each treatment). Malaise was also not observed for chronic or systemic treatments.(C ) Conditioned Taste Aversion expressed as saccharin preference (normalized to control; n=7 each group), showing no illness in BMP7-treated mice. LiCl-treated mice served as a positive control.

0

0.5

1

1.5

2

2.5

3

3.5

control lenti-shRNA

BMPRII lenti-shRNAfo

od c

onsu

mpt

ion

from

mid

nigh

t (g)

afte

r i.c

.v. B

MP

7 *

Supplemental Figure S3

Supplemental Figure S3Mice treated i.c.v. with lentivirus expressing either control shRNA or BMPR2 shRNA. After knock-down by the lentivirus, mice were treated i.c.v with BMP7 for measurement of acute food intake during the dark-phase. Data represent the average of 2 separate experiments, showing that knock-down of central BMPR2 abolishes the anorectic response to i.c.v. BMP7.

Supplemental Table 1: Primer sequences used in Q-RT-PCR

ACAGCCATGGAAATGAGCACAACC

AATGCAAGGATTCACCGAAAGCCC

ALK3 (BMPR1a)

CCGGTCTCCTGTCAACATTCCCCCTGACACAACACCACTCBMPR2

TGAAGGGTTGCTTGTTCTGGGGGCTTACAGCTCTCTGTGGBMP7

TTCACAGTGGTCCTCGTTCCTGCTAATGATGATGGCTTTCCALK2 (ACVR1)

AGCAAAAGGCATTGAAGAAGCTTGTGTTCTGCTGTTGGCACTAgRP

GGGCCCGTCGTCCTTCTCCCGGCCCCAGGAACAGCAGCAGTPOMC

ATTGATGTAGTGTCGCAGAGCGGA

TATCTCTGCTCGTGTGTTTGGGCA

NPY

Reverse Primer Forward PrimerGene Name