Supplementary Data
Supplementary Materials and methods
Primers
Primers used for real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
were as follows: mouse matrix
metalloproteinase (MMP)-10 (forward, 5′-
CCTGTGTTGTCTGTCTCTCCAAGA-3′;
reverse,
CGTGCTGACTGAATCAAAGGAC-3′),
mouse
5
integrin
5′(forward,
5′-
ATATCTGCCAGCGCATCTCTC-3′; reverse, 5′-GCTAAGGTTGATGCAGGACACA3′),
mouse
myocyte
enhancer
factor
(MEF)
GCTTTGTCGTACACTAACCCAGG-3′;
2A
(forward,
reverse,
GCATGCTCGAATCTGCTAATGT-3′),
mouse
MEF2B
5′5′-
(forward,
5′-
TGAAGAAGGCTTATGAGCTGAGC-3′; reverse, 5′-GCGCGCTGTTGAAGATGAT3′), mouse MEF2C (forward, 5′-TCCACCTCCCAGCTTTGAGAT-3′; reverse, 5′TGACAGGATTGCTGTACACCAAAC-3′),
mouse
TCAACCACTCCAACAAGCTGTT-3′;
MEF2D
reverse,
GTACTCGGTGTACTTGAGCAGCA-3′),
mouse
(forward,
5′5′-
glyceraldehyde-3-phosphate
dehydrogenase (GAPDH; forward, 5′-ATGTGTCCGTCGTGGATCTGA-3′; reverse, 5′-
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ATGCCTGCTTCACCACCTTCT-3′),
(forward,
mouse
histone
deacetylases
5′-GGAACTCCTAGCACTGAAACAGAAG-3′;
TTCATGCAACTGTGCCTCATG-3),
mouse
HDAC5
(HDACs)
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reverse,
5′-
(forward,
5′-
TATTCACCATGGCAACGGC-3′; reverse, 5′-CTGGAAAGAAGTTCCCGTTGTC-3′),
mouse
HDAC7
(forward,
5′-AGGAGCAAGAACTTCGGCAA-3′;
TTCTGCTTGACCACACTGCTG-3′),
GCATCTTGCATTCCTTGTGCT-3′;
human
GAPDH
(forward,
human
reverse,
MMP-10
reverse,
(forward,
5′5′-
5′-TTTTGCTGCCCCACTCAGA-3′),
5′-AAATTCCATGGCCACCGTCA-3′;
reverse,
5′-
CATCGCCCCACTTGATTTTG-3′).
The primers utilized for chromatin immunoprecipitation were as follows: mouse
MMP-10 promoter #1 (−87 to −7 bp: forward, 5′-CCCATGTGAGTCATGCTTAGTGA3′; reverse, 5′-CCTGGTTTCCATATCTTCCCTTTA-3′), mouse MMP-10 promoter #2
(−425 to −343 bp: forward, 5′-ACCCAAGGACTCCAGCAATG-3′; reverse, 5′TGGAAAGAGATTGCAAAGTGAATG-3′), negative control locus (−8 kb: forward, 5′TGTCAGAGTTCCCCTTGTGGTT-3′;
reverse,
GGCTCATTGGGTATTGTCGTACT-3′).
Chromatin immunoprecipitation
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5′-
Cells were fixed with 1% formaldehyde for 15 min at room temperature, incubated in
the presence of 125 mM glycine for 5 min and collected in microtubes after washing
with ice-cold phosphate-buffered saline. The cells were further washed twice with
immunoprecipitation buffer [1% Triton X-100, 0.5% NP-40, 50 mM Tris (pH 7.5), 5
mM ethylenediaminetetraacetate (EDTA), 150 mM NaCl] including protease inhibitor
cocktail (Sigma), lysed with sonication buffer [1% sodium dodecyl sulphate (SDS), 50
mM Tris (pH 8.1), 10 mM EDTA, protease inhibitor cocktail] for 10 min on ice and
sonicated on ice for six rounds of 15 s each at the maximum setting of the sonicator
(UR-20P; Tomy Seiko Co., Ltd., Tokyo, Japan). Sheared chromatin was cleared by
centrifugation at 20,000 g for 10 min, and the supernatant was diluted 10 times with
ChIP dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris
(pH 8.1), 150 mM NaCl, protease inhibitor cocktail] and used as chromatin for
immunoprecipitation.
For immunoprecipitation, the chromatin was mixed with normal or specific
antibodies and incubated for 30 min at 4°C in an ultrasonic bath (UC-1331N; Tokyo
Ultrasonic Engineering Co., Ltd., Tokyo, Japan; anti-acetylated H3 and H4 polyclonal
antibodies) or incubated for 16 h at 4°C with rotation (anti-EF2A polyclonal antibody).
Immunocomplexes cleared by centrifugation at 20,000 g for 10 min at 4°C were
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collected by incubating with 20 l of Dynabeads (Invitrogen) for 2 h with rotation. The
beads were then sequentially washed with low salt buffer (0.1% SDS, 1% Triton X-100,
2 mM EDTA, 20 mM Tris (pH 8.1), 150 mM NaCl), high salt buffer (low salt buffer +
350 mM NaCl), LiCl buffer (1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris
(pH 8.1), 250 mM LiCl) and twice with TE (10 mM Tris, 1 mM EDTA) supplemented
with 50 mM NaCl. Thereafter, they were mixed with 10% Chelex 100 (Bio-Rad
Laboratories Inc., Hercules, CA, USA), eluted by boiling for 10 min and treated with 40
g of Proteinase K (Wako) at 55°C for 1 h. After the immunocomplexes were separated
from the beads by brief centrifugation following boiling for 10 min, the amount of DNA
was quantified by real-time RT-PCR and normalized to input.
Smad shRNA expression vectors
To create the shRNA expression lentivirus, oligonucleotides encoding both strands of
the targeting sequence were annealed and ligated with pENTR4 (Invitrogen) entry
vector. Subsequently, shRNA expression cassettes were subcloned into the lentiviral
destination vector CS-RfA, which contained elongation factor 1 promoter-driven
blasticidin S deaminase (CS-RfA-EB) (Katayama et al., 2004; Miyoshi et al., 1998), by
LR recombination reaction (Invitrogen). The target sequences for mouse Smads and the
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non-target shRNA control sequence (5′-CAACAAGATGAAGAGCACCAA-3′) were
obtained from the RNAi consortium shRNA library and Mission shRNA (SigmaAldrich Co., St. Louis, MO, USA), respectively.
To generate a lentivirus expressing shRNA, 293T cells (Riken BRC Cell Bank,
Tsukuba, Japan) were cotransfected with a shRNA expression lentiviral vector and
packaging plasmids (pCAG-HIVgp and pCMV-VSV-G-RSV-Rev) (Nagata et al., 2006)
by calcium phosphate precipitation. The conditioned medium containing viral particles
was harvested at 48 h after transfection and used to infect NMuMG cells with 4 g/ml
polybrene. Successfully infected cells were selected and maintained with 10 g/ml
blasticidine S.
Supplementary Figure legends
Supplementary Figure 1 Effects of cycloheximide and knockdown of Smad proteins on
TGF--mediated MMP-10 induction. (a) NMuMG cells pretreated with 50 ng/ml
cycloheximide (CHX) were stimulated with 2 ng/ml TGF-1 for 24 h, and total RNA
was analysed by real-time RT-PCR. Increase in MMP-10 mRNA is shown as above. (b)
The shRNA expression lentivirus vectors for each Smads were infected into NMuMG
cells, as described in Supplementary Materials and methods, and knockdown effects
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were confirmed by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) is shown as a loading control. (c) Cells expressing shRNA for each Smads
were treated with 2 ng/ml TGF-1 for 24 h. mRNA levels of MMP-10 and integrin 5
were evaluated by real-time RT-PCR as in Figure 2a. *P < 0.05; **P < 0.01. (d) Cells
were transfected with siRNA for Smad3 together with that for MEF2A as described in
Figure 4b, and after the treatment with TGF-, mRNA levels of MMP-10 were
examined as above.
Supplementary Figure 2 (a) Knockdown effects of small interfering RNA (siRNA) for
myocyte enhancer factor (MEF) 2 family transcription factor A. NMuMG cells were
transfected with siRNA for MEF2A or the negative control (Ctr; 100 nM), and after 48
h, they were treated with 2 ng/ml transforming growth factor (TGF)-1 for 24 h. The
expression levels of MEF2A were evaluated by real-time reverse transcriptasepolymerase chain reaction (RT-PCR) (mRNA) as in Figure 2a and by immunoblotting
(protein) as above. GAPDH is shown as a loading control. (b, c) Knockdown effects of
siRNA for MEF2B and D and their effect on TGF--stimulated matrix
metalloproteinase (MMP)-10 induction. NMuMG cells were transfected with siRNA for
MEF2B (b) and MEF2D (c), or a negative control (Ctr; 100 nM), and mRNA levels of
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MEF2B (b) and MEF2D (c) were evaluated together with MMP-10 by RT-PCR as
above. (d) Knockdown effects of siRNA for class IIa histone deacetylases (HDACs).
Effects were verified by real-time RT-PCR as above.
Supplementary Figure 3 (a) Examination of the interaction of MEF2A and Smad
proteins. NMuMG cells expressing the Flag-tagged Smad2, 3 or MEF2A were
established as in Figure 2b and treated with 2 ng/ml TGF-1 for 2 or 24 h. Total
proteins were extracted, subjected to immunoprecipitation with the anti-Flag antibody
and analysed by immunoblotting using specific antibodies as indicated. (b) Knockdown
of Smad3 by lentivirus-mediated shRNA expression and its effect on downregulation of
class IIa HDAC9 by TGF-. Tet-Off cells expressing the Flag-tagged HDAC9 (pSINTRE II system), established as in Figure 4c, were infected by the lentiviruses encoding
shRNA for Smad3, as in Supplementary Figure 1b, and treated with 2 ng/ml TGF-1 for
24 h in the presence or absence of 10 ng/ml Dox. Lysates were subjected to
immunoblotting using the antibody against the Flag tag or Smad3. GAPDH is shown as
a loading control. Quantification of the band densities was performed using Image J
software, and relative intensities are shown.
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