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Supplemental Methods;
Determination of early LV infarct size
Infarct size was determined as described previously (1,2). In brief, 48 hours post-MI,
the heart was quickly excised from anesthetized mice and sliced into five 1.0-mm-thick
sections perpendicular to the long axis of the heart. The sections were then incubated
with 1% triphenyltetrazolium chloride (TTC) (Sigma T8877) in phosphate buffered
saline (pH 7.4) for 15 minutes at room temperature. Pictures were taken under an
inverted Nikon microscope using a 4X lens. Infarct size was determined by measuring
the TTC negative area (infarcted myocardium) and LV circumference by NIS Elements
software. Myocardial infarct size was expressed as a percentage of total LV area.
Determination of myocardial apoptosis
In situ Cell Death Detection kit, TMR red (Roche No. 12156792910) was used for
terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL). The
3-week post-MI heart sections were treated according to the manufacturer’s instructions
and co-stained for cardiomyocyte specific marker Troponin-I and 4,6-diamidino-2phenylindole (DAPI). Slides were applied with mounting media and covered with a glass
cover. A Nikon Eclipse 80i fluorescence microscope was used to visualize the TUNELpositive cardiomyocyte, and NIS Elements software was used to capture the images.
In vivo 5-bromo-2'-deoxyuridine (BrdU) labeling
BrdU (Sigma B5002) was dissolved in 150 mM NaHCO3 to achieve a final
concentration of 5 mg/ml. The solution was passed through a 0.22μm filter and intraperitoneal (i.p.) injected at 50mg/kg body weight twice a day for 5 days during 3rd week
of MI. Post 3 week of MI, hearts were harvested from anesthetized mice and fixed in
1
4% PFA followed by paraffin embedding and sectioning. Immunofluorescence staining
was performed using BrdU labeling and detection kit (Roche 11296736001) as per
manufacturer recommendations.
Sample preparation, immunoblotting
LV lysates were prepared from 3 week post-MI heart as described previously (3).
Protein concentration in the lysate was quantified with the Bicinchoninic acid (BCA)
protein assay (#23225 from Pierce). Equal amounts of proteins were subjected to 420% gradient SDS-PAGE and subsequently were transferred to nitrocellulose
membranes (GE Healthcare Whatman#10401196). Membranes were blocked for 60
minutes using LI-COR blocking buffer. Primary antibody incubations were performed at
different dilutions for different antibodies as described in the antibody list (in
Supplementary data). All incubations for primary antibodies were performed overnight
at 4°C followed by secondary antibody (Alexa Fluor 680 or Alexa Fluor 800 from LICOR) incubation at 1:4000 dilutions for 1 hour at room temperature. After 3X washing,
membranes were scanned with the Odyssey Infrared Imaging System (LI-COR).
Neonatal rat ventricular myocyte isolation
Primary cultures of neonatal rat ventricular myocytes (NRVMs) were prepared from 12-day-old Sprague-Dawley rats as previously described (4). Cells were plated onto
Primaria cell culture dishes (BD) for immunoblotting. Following desired treatments, the
cells were lysed for immunoblotting as described previously.
Immunohistochemistry
Heart sections were deparaffinized with xylene and rehydrated with consecutive
incubations in decreasing concentrations of ethanol and finally incubated in distilled
2
water. For antigen retrieval, specimens were boiled for 20 minutes in preheated citrated
antigen retrieval system (DAKO S1699) using a 920C hot water incubator. After 3
washes with phosphate buffered saline (PBS), endogenous peroxidase activity was
blocked by an additional incubation with 0.15% hydrogen peroxide for 20 minutes at
room temperature followed by 45 min incubation with TNB (blocking) buffer (Perkin
Elmer, Woodbridge, Ontario). Tissue was incubated with primary antibodies for
overnight at 4°C in TNB. After 3 washes in PBS, slides were incubated with secondary
antibodies: EnVisionTM+ System-HRP (DAKO K4002). For fluorescence amplification, a
TRITC-based Tyramide reagent pack from Perkin Elmer was used for Ki67, phosphohistone H3 (Ser10) and cyclin E1 according to the manufacturer instructions. Slides
were then washed 3x with PBS and incubated with cardiomyocyte specific primary
antibody (Sarcomeric α-actinin/ Troponin I) for 60 min at 370C followed by 3x wash with
PBS. Slides were incubated with fluorescence labeled secondary (Alexa fluor488/ Alexa
fluor555) antibodies. After 3X washing with PBS, nuclei were stained by DAPI (1ug/ml)
for 15 min. After a quick PBS wash, tissues were mounted with Vectashield (Vector
laboratories, Burlingame). For Massons’ Trichrome, we followed the manufacturers
instructions for Trichomes Accustain (Masson), Sigma-Aldrich. Pictures were taken
under an inverted Nikon Eclipse 80i fluorescent microscope. Infarct size, fibrosis and
other cell quantifications were performed in a blinded manner using NIS Elements
software.
Cardiomyocyte circumference measurement
Cardiomyocyte cross-sectional area was measured from 5-μm-thick 8 week post-MI
heart sections that had been stained with Massons’ Trichrome using NIS Element
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software. Only myocytes that were round (i.e., were cut in cross section) were included
in the analysis. An observer blind to the genotypes of the heart performed the
measurements.
Quantification of viable cardiomyocytes in scar
Five images from scar area of each Masson trichrome stained sections of post- 8
week MI WT and KO hearts were captured using 20X lens and quantification was
performed using NIS element.
Co-immunoprecipitation
Cells were harvested in RIPA/lysis buffer. After protein quantification, 500 μg of protein
in 500μl lysate was incubated with 2 μg of an antibodies against E2F-1 and cyclin E1
overnight at 4°C using a rotator followed by another 2 hour incubation with Protein G
Plus/ Protein A Agarose beads (Calbiochem#IP05). The beads were washed 3X with
ice cold PBS. Protein complexes were released by 5 min of boiling in SDS sample
buffer, resolved by SDS-PAGE and subjected to immunoblotting.
A detailed list of different antibodies used and applications
S.
Antibody
Vendor
Catalog No
Dilution
Application
1
Beta-Catenin
Cell Signaling
9587
1;1000
Western Blotting
2
p-Beta Catenin (Thr41/Ser45)
Cell Signaling
9565
1;500
Western Blotting
3
GAPDH
Fitzgerald
10R-G109a
1;10,000
Western Blotting
4
GSK-3α/β
Cell Signaling
5676
1;1000
Western Blotting
5
GSK-3α
Cell Signaling
4337
1;1000
Western Blotting
No.
4
6
GSK-3β
Cell Signaling
9315
1;1000
Western Blotting
7
phospho-GSK-3α(Ser21)
Cell Signaling
9316
1;1000
Western Blotting
8
phospho-GSK-3β(Ser9)
Cell Signaling
9336
1;1000
Western Blotting
9
Bax
Cell Signaling
2772
1;1000
Western Blotting
10
Bcl-2
Cell Signaling
2870
1;1000
Western Blotting
11
Cyclin D1
Cell Signaling
2926
1;500
Western Blotting
12
Cdk4
Santa Cruz Biotech
sc-260
1;100
Western Blotting
13
Cdk2
Santa Cruz Biotech
sc-163
1;100
Western Blotting
14
p-Cdk2 (Thr 160)
Cell Signaling
2561
1;500
Western Blotting
15
Retinoblastoma
Cell Signaling
9313
1;500
Western Blotting
16
p-Retinoblastoma (Ser780)
Cell Signaling
3590
1;1000
Western Blotting
17
Cyclin E1
Santa Cruz Biotech
sc-481
1;100
Western Blotting
18
E2F-1
Santa Cruz Biotech
sc-193
1;100
Western Blotting
19
phospho-cyclin E1(Thr395)
Santa Cruz Biotech
sc-12917-R
1;100
Western Blotting
20
α-Smooth muscle actin
Sigma
A5228
1;2000
Western Blotting
21
Vimentin
Cell Signaling
5741
1;1000
Western Blotting
22
Axin-2
Thermo Scientific
PA5-21093
1;500
Western Blotting
23
phospho-NFkB P65 (Ser536)
Cell Signaling
3033
1;500
Western Blotting
24
NFkB P65
Santa Cruz Biotech
Sc-109
1;100
Western Blotting
25
phospho-4E-BP1 (Thr37/46)
Cell Signaling
2855
1;1000
Western Blotting
26
4E-BP1
Cell Signaling
9644
1;1000
Western Blotting
27
p-S6 Ribosomal (Ser235/236)
Cell Signaling
4856
1;1000
Western Blotting
28
S6 Ribosomal protein
Cell Signaling
2317
1;1000
Western Blotting
29
p-P70S6Kinase (Thr389)
Cell Signaling
9205
1;1000
Western Blotting
30
P70S6Kinase
Cell Signaling
2708
1;1000
Western Blotting
5
31
Cyclin E1
Santa Cruz Biotech
sc-481
2μg/500μg
Immunoprecipitation
32
E2F-1
Santa Cruz Biotech
sc-193
2μg/500μg
Immunoprecipitation
33
Ki67
Vector Lab
VP-RM04
1;50
Immunofluorescence
34
Sarcomeric α-actinin
Sigma
A7811
1;200
Immunofluorescence
35
Troponin I
Cell Signaling
4002
1;200
Immunofluorescence
36
phospho-Histone H3 (Ser 10)
Upstate
06-570
1;400
Immunofluorescence
37
Cyclin E1
Abcam
ab52189
1;300
Immunofluorescence
References;
1.
Most P, Seifert H, Gao E et al. Cardiac S100A1 protein levels determine
contractile performance and propensity toward heart failure after myocardial
infarction. Circulation 2006;114:1258-68.
2.
Gao E, Lei YH, Shang X, Huang ZM, Zuo L, Boucher M, Fan Q, Chuprun JK, Ma
XL, Koch WJ. A novel and efficient model of coronary artery ligation and
myocardial infarction in the mouse. Circ Res 2010;107:1445-53.
3.
Lal H, Zhou J, Ahmad F et al. Glycogen synthase kinase-3alpha limits ischemic
injury, cardiac rupture, post-myocardial infarction remodeling and death.
Circulation 2012;125:65-75.
4.
Haq S, Choukroun G, Kang ZB et al. Glycogen synthase kinase-3ß is a negative
regulator of cardiomyocyte hypertrophy. J Cell Biol 2000;151:117-129.
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