0

10

20

30

Avera

ge b

od

y

weig

ht

(g)

Supplemental Figure 1. Remicade treatment did not affect body weight and muscle weight in mdx mice.Mdx mice were treated with Remicade (10 mg/ml) starting either at 2 weeks or 7 months of age for 6 or 7 weeks correspondingly. No significant different was revealed between treated and untreated groups of animals. Vertical bars on all graphs represent standard error. Statistics were assessed by two tailed t-test with significance set at p<0.05.

Body Muscles

Quad TA Gast Hams Tricep Dia Heart0

0.2

0.4

0.6

0.8

Avera

ge m

uscle

weig

ht

(g)

0

10

20

30

40

Avera

ge b

od

y

weig

ht

(g)

9 moBody Muscles

Quad TA Gast Hams Tricep Dia Heart0

0.2

0.4

0.6

0.8

1

1.2

Avera

ge m

uscle

weig

ht

(g)

mdx (n=9) mdx-Remicade (n=10)

mdx (n=5) mdx-Remicade (n=6)

2 mo

Supplemental Figure 2. Echocardiography of 20mg/kg treated mdx mice.Mdx mice were treated with Remicade (20 mg/kg) and control antibody provided by Centocor (20 mg/kg) for 8 months, starting at 6 weeks of age (Exp 3, table 1). Echocardiography was performed at the end of the treatment period. Vertical bars represent standard error. No significant differences were observed in heart rate, left ventricular (LV) mass and ejection fraction (EF), fractional shortening (FS) and ratio between early and late ventricular filling velocity (E/A) as observed by two tailed t-test with significance set at p<0.05

mdx (n=4)

mdx-Remicade (n=5)

0

10

20

30

40

LV

% F

S

0

20

40

60

80

LV

EF

0102030405060708090

LV

Mass

p=0.02

1.6

1.8

2.0

2.2E/A

0

200

400

600H

eart

Rate

(bpm

)

mdx mdx-Remicade mdx-EnbrelB

0

500

1000

1500

2000

A.Summary intensity/muscle volume Average voxel intensity

0.0

10.0

20.0

30.0

40.0

50.0

n=4

n=5

n=5n=4

n=5

n=5

mdx mdx-Enbrelmdx-Remicade

Supplemental Figure 3. Magnetic Resonance Imaging did not reveal improvements following Remicade treatment. Magnetic resonance imaging of the quadriceps muscles of mdx mice injected with Remicade (3 mg/kg), Enbrel (4 mg/kg) or control for Remicade (3 mg/kg) for 6 months starting at age of 6 weeks of age (once weekly dosing). (A) graphs of quantitated MRI data representing calculated intensity per muscle volume, average voxel intensity and muscle volume and muscle weight normalized per body weight; (B) representative T2 maps. Vertical bars represent standard error and statistics were assessed by two tailed t-test with significance set at p<0.05.

320

340

360

380

400

420

440n=4 n=5

n=5

quads TA0.000

0.002

0.004

0.006

0.008

0.010

0.012

B.

Muscle weight/body weightD.C. Muscle Volume

Mdx, n=8

Remicade, n=10

Supplemental Figure. 4: Remicade treatment did not affect abnormal deposits of calcium in diaphragm of mdx mice treated with various doses of the drug. The von Kossa stain was utilized to access mineralization in diaphragm tissue sections of mdx mice treated with Remicade . Shown are representative pictures of the diaphragms of mdx mice treated with Remicade at concentrations 20 mg/kg (A) or 3 mg/kg (B)

mdx mdx-RemicadeA

20 mg/kg

mdx mdx-RemicadeB3 mg/kg

mdx mdx-Enbrelmdx-Remicade

Supplemental Figure 5 Expression of inflammatory genes in diaphragms of mdx mice treated with Remicade (3 mg/kg) and Enbrel (4 mg/kg) for 6 months starting at age of 6 weeks as assessed by real-time PCR. Expression of IL-6, β-TGF, iNOS and osteopontin mRNAs were accessed in diaphragms of mdx mice by quantative PCR. GAPDH PCR is used as cDNA control. Data are shown in graph as average Starting Quantity (SQ) for the gene normalized by SQ for GAPDH. Vertical bars on all graphs represent standard error. Statistics were assessed by two tailed t-test with significance set at p<0.05.

iNO

S/G

AP

DH

iNOS

0

0.5

1

1.5ß

-TG

F/

GA

PD

H

ß-TGF

0

0.5

1

1.5IL

-6/

GA

PD

H

IL-6

0

0.5

1

1.5

oste

op

on

tin

/GA

PD

H

OSTEOPONTIN

0

0.5

1

1.5

p=0.02

diaphragms

mdx

mdx-Enbrel

mdx-Remicade

Supplemental Figure 6: α-Laminin staining of hearts of mdx mice after prolonged treatment (6 months) with Remicade (3 mg/kg) or Enbrel (4 mg/kg).

mdx (n=5)mdx-Enbrel (n=5)

mdx-Remicade (n=7)

mdx (n=6)mdx-Enbrel (n=6)

mdx-Remicade (n=7)

mdx (n=5)mdx-Enbrel (n=6)

mdx-Remicade (n=7)

IGFbp3 IGFbp4 IGFbp50.0

0.5

1.0

1.5

IGFb

p/G

AP

DH

rati

o

IGFbp hearts

p=0.02p=0.01

mdx (n=6)mdx-Enbrel (n=5)

mdx-Remicade (n=7)

0.0

0.5

1.0

1.5

TGF betahearts

TG

Fb

eta

/GA

PD

H r

ati

o

Supplemental Figure 7. Prolonged treatment (6 months) of mdx mice with Remicade (3 mg/kg) or Enbrel (4 mg/kg) decreases myostatin content in the hearts of treated animals while did not significantly affect TGF beta as estimated by quantative PCR. Levels of myostatin, TGF beta and various IGF binding peptides were assessed by quantitative, real time PCR in the hearts of untreated mdx mice compared with Remicade and Enbrel-treated animals. GAPDH PCR is used as a cDNA control. Real-time PCR data is shown in graph as average Starting Quantity (SQ) for the gene normalized by SQ for GAPDH. Vertical bars on all graphs represent standard error. Statistics were assessed by two tailed t-test with significance set at p<0.05.

Phospho-CAMKII

CAMKII

Loading

mdx Remicade Enbrel

Supplemental Figure 8. CAMKII and phospho-CAMKII content in heart lysates of mdx mice after prolonged treatment (6 months) with Remicade (3 mg/kg) or Enbrel (4 mg/kg). Levels of CAMKII and phospho-CAMKII were estimated by Western blotting in the hearts of untreated mdx mice compared with Remicade and Enbrel-treated animals. Specific antibody were utilized to evaluate protein content in heart homogenates prepared in the presence of phosphatase and protease inhibitors. Blots were analyzed by ImageJ software and specific signals were normalized for loading. All values were expressed as percent of average mdx normalized signal. Vertical bars on all graphs represent standard error. Statistics were assessed by two tailed t-test with significance set at p<0.05.

%

CAMKII pCAMKII pCAMKII/CAMKII

0

50

100

150

hearts