Supplementary Methods
Animals
All animal studies were approved by The Animal Care and Use Committee of TelAviv Sourasky Medical Center, which conforms to the policies of the American Heart
Association and the Guide for the Care and Use of Laboratory Animals.
For real-time PCR and in-situ hybridization, C57BL/6 female mice aged 8 weeks
were purchased from Harlan Laboratories, Israel. MiR-106b~25 knockout mice were
obtained by crossing of miR-106b~25-/+ mice. Wild-type littermates were used as
controls.
Hindlimb Ischemia Model and Laser-Doppler Perfusion Imaging
Female 8 weeks old mice were anesthetized with 2% isoflurane. The femoral artery
was exposed by incision of the skin at the middle portion of the left hindlimb. Both
the proximal and the distal ends of the femoral artery were ligated and the artery was
excised. Shortly after surgery, as well as 7 and 14 days postoperatively, mice were
placed on a heating pad for several minutes and the blood flow in both hindlimbs was
determined by laser-Doppler perfusion imaging (LDPI).Color-coded images were
obtained with Moor laser Doppler imager (LDI)scanner (Moor Instruments,
Axminster, UK). For each mouse, perfusion was calculated by the Moor LDI software
as a percentage of perfusion in the non-ischemic limb.
RNA Extraction and Quantitative Real-Time PCR
Total RNA was extracted with a phenol/chloroform EZRNA kit (Biological
Industries, Beit Ha-Emek, Israel). For miRNA detection, 25ng of total RNA was
transcribed to cDNA using the universal RT LNATM cDNA Synthesis Kit (Exiqon,
Vedbaek, Denmark). Quantitative real-time PCR was performed with Sybr Green and
specific locked nucleic acid (LNA) primers for miR-106b, miR-93 and miR-25
(Exiqon, Denmark), using a StepOnePlus instrument (Applied Biosystems). Results
were derived by the Comparative CT (ΔΔCt) method.
In-Situ Hybridization
To detect expression of miR-93 and miR-25, skeletal muscle was processed and
stained according to the method of Obernosterer 30 with several modifications. Briefly,
tissue was fixed overnight with 4% formalin, transferred overnight into phosphatebuffered saline (PBS) containing 30% sucrose, then embedded in OCT and frozen on
dry ice. Sections (10 µM thick) were cut and thawed for 30 minutes at room
temperature. The sections were fixed with 4% paraformaldehyde for 10 minutes,
incubated with 1µg/mL ProteinaseK(Sigma-Aldrich, St. Louis, MO) for 8 minutes,
and prehybridized with hybridization buffer for 12 hours at 55oC. Double-DIGlabeled LNA ISH probes for miR-25or mir-93 (Exiqon, Denmark) were added to the
sections and kept overnight in a Dako hybridizer. A U6 probe was used as a positive
control and a scramble probe as a negative control. Anti-DIG antibody (1:100;Roche)
was added for 1hour. NBT/BCIP substrates (Roche) were added to the slides and
counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA).
Plasmids
The MDH1-106b~25 and MDH1-106b~25 plasmids were used for in-vivo injections.
For retroviral transductions, the plasmids used were pMSCV-miR25, pMSCVmiR106b, pMSCV-miR-93, and pMSCV-HTR.
Cell Culture
Bone marrow cells from tibias and femur bones were flushed with PBS of mir106b~25 wild-type and KO mice. Mononuclear cells were separated by density
gradient centrifugation (Axis-Shield, Dundee, Scotland) and grown on 6-well tissue
culture dishes (Nunc,Denmark).Non-adherent cells were washed out after 3 days.
Cells were maintained for 16 days at 37oC, 5% CO2 in DMEM supplemented with
15% fetal calf serum (FCS), 1% penicillin/streptomycin, and 1% glutamine (all from
Biological Industries). Passage 3-4 cells were used for all experiments. Murine H5V
endothelial cells (a murine-immortalized heart-derived endothelial cell line) were
cultured in DMEM/F12 supplemented with 10% FCS, 1% penicillin/streptomycin,
and 1% glutamine.
Migration Assay
VEGF(10 ng/mL) or SDF1 (Peprotech, Rehovot, Israel) was loaded into the lower
well of a transwell insert with 8-µpore filters (Nunc, Denmark). Bone marrow cells
(2×105 cells) were loaded into the upper well and incubated for 4 hours at 37°C in a
humidified chamber. Medium from the lower well was collected and the number of
migrating cells was counted for 30seconds in a BD Biosciences FACSCanto II flow
cytometer. For PTEN inhibition, cells were treated with VO-OHpic trihydrate (SantaCruz) at 1µM for 24 hours.
Proliferation assay
Cells (2×104 cells) were plated in 96-well plates and incubated overnight in DMEM
serum-free medium.FCS (20%) was then added to the medium for 16hours.
Proliferation was assayed with the XTT Cell Proliferation Assay Kit (Biological
Industries) according to the manufacturer’s instructions.
In-Vitro Matrigel Tube-Formation Assay
For this assay, 2×105 BMSCs, or 105 BMSCs mixed with 105 H5V cells, or 2×105
H5V cells mixed with 200µL of medium conditioned by bone marrow cells either
from miR-106b~25wild-type mice or from miR-106b~25 KO mice, were seeded in
each well of 24-well plates coated with 200μL Matrigel (BD Biosciences). For PTEN
inhibition, cells were treated with VO-OHpic trihydrate (Santa-Cruz) at 1µM for 24
hours.
Cumulative tube length was measured using the NIS-Elements software (v3.0) on 3
random images (X4). Conditioned medium was obtained by adding 1.5mL of serumfree DMEM to 1.5×106bone marrow cells for 24hours and then centrifuging at 300×g
for 5minutes to remove cell debris.
Flow Cytometry
Bone marrow cells were incubated with anti PE-Sca-1, anti-APC-FLK-1 and
antiFITC-C-kit antibodies (eBioscience, San Diego, CA) or their corresponding
isotype controls, for 30minutes at 4oC in the dark. Cells were analyzed in a flow
cytometer (BD Biosciences FACSCanto II). The results were analyzed by FACSDiva
software (all from Becton Dickinson, Franklin Lakes, NJ).
Mouse angiogenesis cytokine array
For analysis of BMSC conditioned media, Proteome Profiler was used (R&D
Systems, Minneapolis, MN) according to the manufacturer’s instructions.
Conditioned medium (200 µL) was obtained 1.5×106 BMSCs from Mir-106b~25 wild
type or KO mice. Cells were grown overnight in a hypoxic chamber (1% O2). Media
were incubated with antibody cocktails, and then with arrayed antibody membranes.
Signals were detected using labeled strepavidine. Data were analyzed using ImageJ
software.
Retroviral Infection
pMSCV-miR retroviral plasmids were transfected into Phoenix packaging cells
(American Type Culture Collection (ATCC), Manassas, VA;http:// www.atcc.org)
using the standard Ca2PO4 protocol. Media were changed after 24 hours. Cells were
grown for a further 24 hours and virus was then collected and centrifuged at 1500 rpm
at 4°Cfor 5 minutes. H5V endothelial cells were incubated with viral supernatant for
24 hours in the presence of 5 µg/ml polybrene (Sigma-Aldrich).
ELISA for Cleaved Caspase-3
Wild-type or KO BMSCs were exposed for 24h to 400µM H2O2. Cells were then
collected and lysed with lysis buffer. Cleaved caspase-3 was assayed, in 40µg of
protein, using a commercially available Cleaved Caspase-3 kit (R&D Systems)
according to the manufacturer's instructions.
Myocardial infarction model
Animals were anesthetized with ketamine and xylazine. Animals were orally
intubated with 22 GIV catheter and artificially ventilated with a respirator (Harvard
Apparatus). A small oblique thoracotomy was performed lateral to the left intercostal
line in the third costal space to expose the heart. After the pericardium was opened,
the proximal left anterior descending artery (LAD) branch of the left coronary artery
was ligated 6-0 polypropylene sutures through a dissecting microscope. The left
ventricle of the heart was dissected 3 days after the induction of MI intro infarct area
and remote area (healthy tissue adjacent to the infarct), expression of miRNAs was
compared to left ventricles of sham operated animals.
Elisa for PTEN
293Hek cells were transfected with 2µg of either MDH1-106b~25 or MDH1-GFP
plasmids using lipofectamine2000 (Invitrogen).PTEN was assayed after 48 hours, in
80µg of protein, using a commercially available PTEN elisa kit (Cell signaling)
according to the manufacturer's instructions.
Western blot
Cells were washed with phosphate-buffered saline (PBS) and lysed with lysis buffer
A (50 mM Tris–HCl pH 7.6, 20 mM MgCl2, 200 mM NaCl, 0.5% Igepal® CA-630
(Sigma), 1 mM DTT, and antiproteases). Lysates were subjected to polyacrylamide
gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS),
followed by immunoblotting with one of the following Abs: anti-AKT 1:1000, antiphosphorylated-AKT 1:1000 (Cell Signaling), anti-GAPDH 1:4000.Immunoblots
were then exposed to peroxidase-conjugated goat anti-mouse IgG, peroxidaseconjugated goat anti-rabbit IgG, (all at 1:2500), and protein bands were visualized
with the ECL kit. Protein bands were quantified by densitometry with the ImageJ
software.
Aortic rings angiogenesis assay
Aortic rings angiogenesis assay was performed according to the method of Baker20.
Briefly, aortas were dissected from Mir-106b~25 wild-type or KO mice and fat was
gently removed with tweezers. Aortas were cut into small pieces of approximately
0.5mm and placed on a tissue culture plate with Optim-MEM (Gibco) without serum.
After over-night incubation aortic rings were imbedded in Matrigel (BD) and fed with
Opti-MEM supplemented with 2.5% FBS and 30ng/ml of VEGF (peprotech). At day
3 the hypoxia group was treated with 1% hypoxia for 10 hours. The number of
sprouting capillaries was counted at day 6.