Supplemental Figure S1: MicroRNA-486 is a muscle-enriched microRNA whose expression is reduced in dystrophin-deficient muscle.

Supplemental Figure S1

02468

1012141618

Rel

ativ

e ex

pres

sion

/RN

U6-

2 Human miR-486-5pA.

B.

C.

0

2

4

6

8

10

12

Rel

ativ

e ex

pres

sion

/RN

U6-

2 Mouse miR-486-5p

0.00.20.40.60.81.01.21.41.61.82.0

Rel

ativ

e fo

ld e

xpre

ssio

n/R

NU

6-2 Mouse miR-486-5p WT

mdx5cv

***

** ** **NS

Supplemental Figure S2

Supplemental Figure S2: MicroRNA-486 overexpressing mice have improved gastrocnemius mdx5cv muscle histology.

mdx5cv:miR-486 Tgmdx5cv

A.WT miR-486 Tg

Gas

troc

nem

ius

B. Gastrocnemius

0

10

20

30

40

50

60

70

80

90

100

mdx5cv mdx5cv:miR-486 Tg

% o

f Fib

ers/

600

Fibe

rsPercent Fibers with Centralized Nuclei

*

Adult Mice: 2-4 months

Supplemental Figure S3: Skeletal muscle fiber type is not significantly altered in miR-486 overexpressing mice.

Supplemental Figure S3Adult Mice: 2-4 monthsA.

B.

0102030405060708090

100

Type I Type IIa Type IIb

% F

iber

Typ

e

Soleus WTmiR-486 Tgmdx5cvmdx5cv:miR-486 Tg

0102030405060708090

100

Type I Type IIa Type IIb

% F

iber

Typ

e

TA

0102030405060708090

100

Type I Type IIa Type IIb

% F

iber

Typ

e

Gastroc

****

****

**

**

WT

Type

IIa/

Type

I

miR-486 Tg mdx5cv mdx5cv:miR-486 Tg

WT miR-486 Tg mdx5cv mdx5cv:miR-486 Tg

Type

IIa/

Type

I

Sol

TA

Supplemental Figure S4: Serum dystrophic biochemistries are improved in aged dystrophic mice.

Supplemental Figure S4

B.A.

0500

1000150020002500300035004000

Enzy

mat

ic U

/µl S

erum

Serum CK

0

50

100

150

200

Enzy

mat

ic U

/µl S

erum

Serum ALT

NS

NS

Aged Mice: 10-14 months

****

GAPDH

MyHC

50%DM (2% FBS)GM (20% FBS)

D1 D2 D3 D4 D7

DOCK3

Dp427

Normal DMD

90% 50%DM (2% FBS)GM (20% FBS)

D1 D2 D3 D4 D790%

A. B.

0

2

4

6

8

10

12

14

16

50% confl.

90% confl.

Day 1 diff.

Day 2 diff.

Day 3 diff.

Day 4 diff.

Day 7 diff.

Rel

ativ

e ex

pres

sion

leve

l/18s

Rb DOCK3 qPCR Normal

DMD

ND ND

*

****

Supplemental Figure S5

Supplemental Figure S5: DOCK3 expression increases during myogenic differentiation and is highly expressed in DMD myotubes.

C.

0

2

4

6

8

10

0 1 3 5 7 14

Rel

ativ

e Ex

pres

sion

/18s

Rb

Days Post TA Ctx Injury

Dock3 qPCR WTmdx5cv

**

***

**

Supplemental Figure S6

Supplemental Figure S6: Overexpression of miR-486 in dystrophic myotubes increases activated RAC1 expression levels.

Primary Myotubes

scram

bled-m

iR

miR-48

6

scram

bled-m

iR

miR-48

6

Normal DMD

RHOA

RAC1 (Total)

β-actin

RAC1-GTP

DOCK3

CDC42

Primary Antisera

Supplemental Table S1: List of primary antibodies used in this manuscript.

Supplemental Table S1

Antibody Company Species Use/Dilutionβ-tubulin-HRP Cell Signaling Technology Rabbit monoclonal WB: 1:2000β-actin (AC-15) Sigma-Aldrich Mouse monoclonal WB: 1:2000

GAPDH (FL-335) Santa Cruz Biotechnologies Rabbit polyclonal WB: 1:5000PTEN Cell Signaling Technology Rabbit monoclonal WB: 1:1000

phospho-AKT (S473) Cell Signaling Technology Rabbit monoclonal WB: 1:1000phospho-AKT (T308) Cell Signaling Technology Rabbit monoclonal WB: 1:1000AKT (pan) (C67E7) Cell Signaling Technology Rabbit monoclonal WB: 1:1000

AKT1 Cell Signaling Technology Rabbit monoclonal WB: 1:1000AKT2 Cell Signaling Technology Rabbit monoclonal WB: 1:1000AKT3 Cell Signaling Technology Rabbit monoclonal WB: 1:1000

DOCK3/MOCA (H-93) Santa Cruz Biotechnologies Rabbit polyclonal WB: 1:1000Hemagglutinin/HA Clontech Laboratories, Inc Rabbit polyclonal WB: 1:1000

Cleaved Caspase-3 (5A1E) Cell Signaling Technology Rabbit monoclonal WB: 1:1000Rac1 ThermoScientific Mouse monoclonal WB: 1:1000

RhoA (67B9) Cell Signaling Technology Rabbit monoclonal WB: 1:1000Cdc42 (11A11) Cell Signaling Technology Rabbit monoclonal WB: 1:1000

Dystrophin (C-term) Abcam Rabbit polyclonal WB: 1:1000Myosin Heavy Chain Maine Biotechnology Mouse monoclonal WB: 1:1000Laminin α2 (4H8-2) Sigma-Aldrich Rat monoclonal IF: 1:200

Laminin (L9393) Sigma-Aldrich Rabbit polyclonal IF: 1:200MF20 (myosin heavy chain) DSHB Iowa Mouse monoclonal IF: 1:50BA-F8 (Type I, slow myosin) DSHB Iowa Mouse monoclonal IF: 1:200

SC-71 (Type IIa myosin) DSHB Iowa Mouse monoclonal IF: 1:50BF-F3 (Type IIb myosin) DSHB Iowa Mouse monoclonal IF: 1:50

Supplemental Table S2Real Time qPCR Primers

Primer Name Gene Symbol Sequence 5' to 3' SpecieshDOCK3_qPCR_forward1 DOCK3 CAGGTGCGGGAGGTTAAGC HumanhDOCK3_qPCR_reverse1 DOCK3 CTGAGACACTAATCTGGTCCGA Humanh18sRib_qPCR_forward1 18sRib CCGATAACGAACGAGACTCTGG Humanh18sRib_qPCR_reverse1 18sRib TAGGGTAGGCACACGCTGAGCC HumanmDock3_qPCR_forward1 Dock3 AGAGGAGTTTCAACAAAGAAGCC MousemDock3_qPCR_reverse1 Dock3 CAGACTGGCCCACTCTTGTAG Mouse18sRib_qPCR_forward1 18sRib CTCAACACGGGAAACCTCAC Mouse18sRib_qPCR_reverse1 18sRib TGCCAGAGTCTCGTTCGTTAT Mouse

Supplemental Table S2: List of real time PCR primers used in this manuscript.