msa miR486Tgmdx Supplemental Figure S1 v1 - cloudfront.net
Supplemental Figure S1 Relative expression/RNU6-2
A.
Relative expression/RNU6-2
B.
18 16 14 12 10 8 6 4 2 0
Human miR-486-5p
12
Mouse miR-486-5p
10 8 6 4 2 0
Relative fold expression/RNU6-2
C. 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
WT
Mouse miR-486-5p
*
*
**
mdx5cv
NS
**
**
*
Supplemental Figure S1: MicroRNA-486 is a muscle-enriched microRNA whose expression is reduced in dystrophin-deficient muscle.
Supplemental Figure S2 Adult Mice: 2-4 months
A.
miR-486 Tg
mdx5cv
mdx5cv:miR-486 Tg
Gastrocnemius
WT
B.
Gastrocnemius 100
Percent Fibers with Centralized Nuclei
% of Fibers/600 Fibers
90
*
80 70 60 50 40 30 20 10 0
mdx5cv
mdx5cv:miR-486 Tg
Supplemental Figure S2: MicroRNA-486 overexpressing mice have improved gastrocnemius mdx5cv muscle histology.
Supplemental Figure S3 Adult Mice: 2-4 months
A.
WT
Soleus
miR-486 Tg
100 90 80 70 60 50 40 30 20 10 0
% Fiber Type
**
**
**
mdx5cv
**
Type I
mdx5cv:miR-486 Tg
Type IIa
Type IIb
TA 100 90 80 70 60 50 40 30 20 10 0
% Fiber Type
** **
Type I
Type IIa
Type IIb
% Fiber Type
Gastroc
B.
100 90 80 70 60 50 40 30 20 10 0
Type I
miR-486 Tg
Type IIb
mdx5cv
Type IIa/Type I
WT
Type IIa
Sol WT
Type IIa/Type I
mdx5cv:miR-486 Tg
miR-486 Tg
mdx5cv
mdx5cv:miR-486 Tg
TA
Supplemental Figure S3: Skeletal muscle fiber type is not significantly altered in miR-486 overexpressing mice.
Supplemental Figure S4 Aged Mice: 10-14 months A.
B. 3500
**
3000 2500 2000 1500 1000 500 0
200
Serum CK
NS
Enzymatic U/µl Serum
Enzymatic U/µl Serum
4000
Serum ALT
**
150 100
NS
50 0
Supplemental Figure S4: Serum dystrophic biochemistries are improved in aged dystrophic mice.
Supplemental Figure S5
16 Relative expression level/18sRb
A.
DOCK3 qPCR
Normal
14
DMD
12
*
10
50%
90%
DMD GM (20% FBS)
DM (2% FBS) D1
D2
D3
D4
D7
50%
90%
DM (2% FBS) D1
D2
D3
D4
DOCK3
6
**
4 2
ND
0
Relative Expression/18sRb
Normal GM (20% FBS)
8
C.
B.
*
*
Dp427 MyHC
ND
50% 90% Day 1 Day 2 Day 3 Day 4 Day 7 confl. confl. diff. diff. diff. diff. diff.
10
6 4
*
2 0
WT
Dock3 qPCR
8
0
**
**
*
GAPDH
mdx5cv
*
1 3 5 7 Days Post TA Ctx Injury
14
Supplemental Figure S5: DOCK3 expression increases during myogenic differentiation and is highly expressed in DMD myotubes.
D7
m
sc ra m
bl ed -m iR -4 iR 86 sc ra m bl ed m iR -m -4 iR 86
Supplemental Figure S6
Normal
DMD RAC1-GTP RAC1 (Total) RHOA CDC42 DOCK3
β-actin Primary Myotubes
Supplemental Figure S6: Overexpression of miR-486 in dystrophic myotubes increases activated RAC1 expression levels.
Supplemental Table S1 Primary Antisera Antibody β-tubulin-HRP β-actin (AC-15) GAPDH (FL-335) PTEN phospho-AKT (S473) phospho-AKT (T308) AKT (pan) (C67E7) AKT1 AKT2 AKT3 DOCK3/MOCA (H-93) Hemagglutinin/HA Cleaved Caspase-3 (5A1E) Rac1 RhoA (67B9) Cdc42 (11A11) Dystrophin (C-term) Myosin Heavy Chain Laminin α2 (4H8-2) Laminin (L9393) MF20 (myosin heavy chain) BA-F8 (Type I, slow myosin) SC-71 (Type IIa myosin) BF-F3 (Type IIb myosin)
Company Cell Signaling Technology Sigma-Aldrich Santa Cruz Biotechnologies Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Santa Cruz Biotechnologies Clontech Laboratories, Inc Cell Signaling Technology ThermoScientific Cell Signaling Technology Cell Signaling Technology Abcam Maine Biotechnology Sigma-Aldrich Sigma-Aldrich DSHB Iowa DSHB Iowa DSHB Iowa DSHB Iowa
Species Rabbit monoclonal Mouse monoclonal Rabbit polyclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Mouse monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit polyclonal Mouse monoclonal Rat monoclonal Rabbit polyclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal
Supplemental Table S1: List of primary antibodies used in this manuscript.
Use/Dilution WB: 1:2000 WB: 1:2000 WB: 1:5000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 IF: 1:200 IF: 1:200 IF: 1:50 IF: 1:200 IF: 1:50 IF: 1:50
Supplemental Table S2
Real Time qPCR Primers
Primer Name Gene Symbol hDOCK3_qPCR_forward1 DOCK3 hDOCK3_qPCR_reverse1 DOCK3 h18sRib_qPCR_forward1 18sRib h18sRib_qPCR_reverse1 18sRib mDock3_qPCR_forward1 Dock3 mDock3_qPCR_reverse1 Dock3 18sRib_qPCR_forward1 18sRib 18sRib_qPCR_reverse1 18sRib
Sequence 5' to 3' CAGGTGCGGGAGGTTAAGC CTGAGACACTAATCTGGTCCGA CCGATAACGAACGAGACTCTGG TAGGGTAGGCACACGCTGAGCC AGAGGAGTTTCAACAAAGAAGCC CAGACTGGCCCACTCTTGTAG CTCAACACGGGAAACCTCAC TGCCAGAGTCTCGTTCGTTAT
Supplemental Table S2: List of real time PCR primers used in this manuscript.
Species Human Human Human Human Mouse Mouse Mouse Mouse
A.
Relative expression/RNU6-2
B.
18 16 14 12 10 8 6 4 2 0
Human miR-486-5p
12
Mouse miR-486-5p
10 8 6 4 2 0
Relative fold expression/RNU6-2
C. 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
WT
Mouse miR-486-5p
*
*
**
mdx5cv
NS
**
**
*
Supplemental Figure S1: MicroRNA-486 is a muscle-enriched microRNA whose expression is reduced in dystrophin-deficient muscle.
Supplemental Figure S2 Adult Mice: 2-4 months
A.
miR-486 Tg
mdx5cv
mdx5cv:miR-486 Tg
Gastrocnemius
WT
B.
Gastrocnemius 100
Percent Fibers with Centralized Nuclei
% of Fibers/600 Fibers
90
*
80 70 60 50 40 30 20 10 0
mdx5cv
mdx5cv:miR-486 Tg
Supplemental Figure S2: MicroRNA-486 overexpressing mice have improved gastrocnemius mdx5cv muscle histology.
Supplemental Figure S3 Adult Mice: 2-4 months
A.
WT
Soleus
miR-486 Tg
100 90 80 70 60 50 40 30 20 10 0
% Fiber Type
**
**
**
mdx5cv
**
Type I
mdx5cv:miR-486 Tg
Type IIa
Type IIb
TA 100 90 80 70 60 50 40 30 20 10 0
% Fiber Type
** **
Type I
Type IIa
Type IIb
% Fiber Type
Gastroc
B.
100 90 80 70 60 50 40 30 20 10 0
Type I
miR-486 Tg
Type IIb
mdx5cv
Type IIa/Type I
WT
Type IIa
Sol WT
Type IIa/Type I
mdx5cv:miR-486 Tg
miR-486 Tg
mdx5cv
mdx5cv:miR-486 Tg
TA
Supplemental Figure S3: Skeletal muscle fiber type is not significantly altered in miR-486 overexpressing mice.
Supplemental Figure S4 Aged Mice: 10-14 months A.
B. 3500
**
3000 2500 2000 1500 1000 500 0
200
Serum CK
NS
Enzymatic U/µl Serum
Enzymatic U/µl Serum
4000
Serum ALT
**
150 100
NS
50 0
Supplemental Figure S4: Serum dystrophic biochemistries are improved in aged dystrophic mice.
Supplemental Figure S5
16 Relative expression level/18sRb
A.
DOCK3 qPCR
Normal
14
DMD
12
*
10
50%
90%
DMD GM (20% FBS)
DM (2% FBS) D1
D2
D3
D4
D7
50%
90%
DM (2% FBS) D1
D2
D3
D4
DOCK3
6
**
4 2
ND
0
Relative Expression/18sRb
Normal GM (20% FBS)
8
C.
B.
*
*
Dp427 MyHC
ND
50% 90% Day 1 Day 2 Day 3 Day 4 Day 7 confl. confl. diff. diff. diff. diff. diff.
10
6 4
*
2 0
WT
Dock3 qPCR
8
0
**
**
*
GAPDH
mdx5cv
*
1 3 5 7 Days Post TA Ctx Injury
14
Supplemental Figure S5: DOCK3 expression increases during myogenic differentiation and is highly expressed in DMD myotubes.
D7
m
sc ra m
bl ed -m iR -4 iR 86 sc ra m bl ed m iR -m -4 iR 86
Supplemental Figure S6
Normal
DMD RAC1-GTP RAC1 (Total) RHOA CDC42 DOCK3
β-actin Primary Myotubes
Supplemental Figure S6: Overexpression of miR-486 in dystrophic myotubes increases activated RAC1 expression levels.
Supplemental Table S1 Primary Antisera Antibody β-tubulin-HRP β-actin (AC-15) GAPDH (FL-335) PTEN phospho-AKT (S473) phospho-AKT (T308) AKT (pan) (C67E7) AKT1 AKT2 AKT3 DOCK3/MOCA (H-93) Hemagglutinin/HA Cleaved Caspase-3 (5A1E) Rac1 RhoA (67B9) Cdc42 (11A11) Dystrophin (C-term) Myosin Heavy Chain Laminin α2 (4H8-2) Laminin (L9393) MF20 (myosin heavy chain) BA-F8 (Type I, slow myosin) SC-71 (Type IIa myosin) BF-F3 (Type IIb myosin)
Company Cell Signaling Technology Sigma-Aldrich Santa Cruz Biotechnologies Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Cell Signaling Technology Santa Cruz Biotechnologies Clontech Laboratories, Inc Cell Signaling Technology ThermoScientific Cell Signaling Technology Cell Signaling Technology Abcam Maine Biotechnology Sigma-Aldrich Sigma-Aldrich DSHB Iowa DSHB Iowa DSHB Iowa DSHB Iowa
Species Rabbit monoclonal Mouse monoclonal Rabbit polyclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Mouse monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit polyclonal Mouse monoclonal Rat monoclonal Rabbit polyclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal
Supplemental Table S1: List of primary antibodies used in this manuscript.
Use/Dilution WB: 1:2000 WB: 1:2000 WB: 1:5000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 WB: 1:1000 IF: 1:200 IF: 1:200 IF: 1:50 IF: 1:200 IF: 1:50 IF: 1:50
Supplemental Table S2
Real Time qPCR Primers
Primer Name Gene Symbol hDOCK3_qPCR_forward1 DOCK3 hDOCK3_qPCR_reverse1 DOCK3 h18sRib_qPCR_forward1 18sRib h18sRib_qPCR_reverse1 18sRib mDock3_qPCR_forward1 Dock3 mDock3_qPCR_reverse1 Dock3 18sRib_qPCR_forward1 18sRib 18sRib_qPCR_reverse1 18sRib
Sequence 5' to 3' CAGGTGCGGGAGGTTAAGC CTGAGACACTAATCTGGTCCGA CCGATAACGAACGAGACTCTGG TAGGGTAGGCACACGCTGAGCC AGAGGAGTTTCAACAAAGAAGCC CAGACTGGCCCACTCTTGTAG CTCAACACGGGAAACCTCAC TGCCAGAGTCTCGTTCGTTAT
Supplemental Table S2: List of real time PCR primers used in this manuscript.
Species Human Human Human Human Mouse Mouse Mouse Mouse
