1. Volume 11, Issue 1, Pages 93-104 (July 2006)
δEF1 Mediates TGF-β Signaling in Vascular Smooth Muscle Cell Differentiation 
Go Nishimura, Ichiro Manabe, Kensuke Tsushima, Katsuhito Fujiu, Yumiko Oishi, Yasushi Imai, Koji Maemura, Makoto Miyagishi, Yujiro Higashi, Hisato Kondoh, Ryozo Nagai 
Developmental Cell 
Volume 11, Issue 1, Pages (July 2006)
DOI: /j.devcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 δEF1 Expression in Tissues and during SMC Differentiation
(A) Northern blot analysis of tissue distribution of δEF1 mRNA in adult rats.
(B) δEF1 expression in the cardiovascular system. δEF1 knockout mice harbor a LacZ reporter gene controlled by the δEF1 regulatory program (Takagi et al., 1998). Tissues taken from adult δEF1+/− mice were subjected to X-gal staining and counterstained with nuclear fast red. Cross-sections of the thoracic aorta (Ba) and femoral artery (Bb) are shown. Scale bars, 50 μm.
(C) δEF1 expression during SMC differentiation of A404 cells. Expression of δEF1, SM α-actin, and SM-MHC was analyzed by semiquantitative multiplex PCR with primer sets for the gene of interest and an internal control (18S rRNA).
(D) Real-time PCR analysis of δEF1 expression in the aortas of embryos (E18.5) and adult mice. The relative numbers of δEF1 transcripts were normalized to those of 18S. Bars indicate relative copy number and SE.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2 δEF1 Transactivated SMC Differentiation Marker Gene Promoters
(A) Effects of δEF1 on the regulatory region of SMC differentiation marker genes. Reporter genes driven by the regulatory region of indicated genes were transiently cotransfected with the δEF1 expression vector or an empty vector. The β-galactosidase activity of each reporter construct cotransfected with δEF1 expression vector was normalized to that cotransfected with the empty vector. Bars indicate relative β-galactosidase activity and SE.
(B–E) Effect of δEF1 on expression of endogenous SMC differentiation marker genes. Cultured SMCs were infected with either the δEF1 adenovirus or empty adenovirus at 20 MOI and then harvested 36 hr after infection. mRNA expression was analyzed by real-time RT-PCR. δEF1 detected in (B) included transcripts from both endogenous and exogenous genes. The copy number of each transcript was normalized to that of 18S, after which the expression was further normalized to that of cells infected with empty adenoviral vector. Bars indicate relative copy number and SE.
(F–I) Effects of δEF1 knockdown on SMC differentiation of A404 cells. A404 cells were transfected with either δEF1-siRNA or scrambled-siRNA. Twenty-four hours after transfection, the cells were treated with all-trans retinoic acid. Two and 4 days after the initiation of differentiation, the cells were harvested and subjected to Western analysis of δEF1 (F) and real-time PCR analysis of δEF1 (G), SM α-actin (H), and SM-MHC (I). Bars indicate relative copy number and SE.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Analysis of δEF1-Mediated Transactivation of SM α-actin Promoter
(A) Schematic representation of the transcriptional regulatory regions of SM α-actin.
(B) Effects of δEF1 on the transcriptional activity of SM α-actin deletion mutants. Luciferase reporter constructs containing the indicated SM α-actin deletion mutants were transiently cotransfected into cultured SMCs along with either δEF1 expression vector or empty vector. Bars indicate relative luciferase activity and SE; ∗p < 0.05.
(C) Effects of mutations within the cis-regulatory elements on transactivation of the SM α-actin promoter (−271/+42) by δEF1. THR, CArGs, TCE, and E boxes were mutated within the −271/+42 bp construct. The luciferase activity of each reporter construct cotransfected with the δEF1 vector was normalized to the activity of the reporter cotransfected with empty vector. Effects of the mutations on the basal SM α-actin promoter activity are shown in Figure S5. Bars indicate relative luciferase activity and SE.
(D) EMSA analysis of the binding of δEF1 to the SM α-actin promoter. 32P-labeled wild-type THR (wt), mutant THR (mut), E1, and E2 oligonucleotides were incubated with SMC nuclear extracts and subjected to EMSA. In lanes 2–7, a molar excess of the indicated cold competitor was added to the reactants; in lanes 8 and 9, anti-δEF1 or anti-USF antibody was added. Asterisks indicate nonspecific shift bands.
(E) Binding of SMC transcription factors to CArG-B. Nuclear extracts of cultured SMCs were incubated with biotinylated wild-type or mutant CArG-B probe, and the probe and bound proteins were collected with streptavidin-conjugated magnet beads. The bound proteins were subjected to SDS-PAGE and immunoblotting with anti-SRF, anti-δEF1, and anti-Smad3 antibodies.
(F) Binding of in vitro translated proteins to the CArG-B probe. SRF and/or δEF1 were incubated with the biotinylated wild-type CArG-B probe. Input proteins (1 and 2) and bound proteins (3 and 4) were immunoblotted with anti-SRF (1 and 3) or anti-δEF1 antibody (2 and 4).
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 4 Interaction between δEF1 and SRF
(A and B) Physical association of δEF1 and SRF. Whole-cell lysates prepared from cultured rat aortic SMCs were incubated with anti-δEF1 antibody (A), anti-SRF antibody (B), or normal IgG (control). The reactants were immunoprecipitated with protein G (A) or anti-rabbit IgG (B) agarose-conjugated antibody, and immunoprecipitates were subjected to immunoblotting with anti-SRF antibody (A) or anti-δEF1 antibody (B).
(C and D) Analysis of the interaction between domains within δEF1 and SRF proteins. Myc-tagged δEF1 or SRF deletion constructs were transfected into cultured SMCs. Lysates of the cells were incubated with the indicated antibodies (IP), and immunoprecipitates were subjected to immunoblot with the indicated antibodies (IB).
(E) The domains are schematically illustrated.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 5 δEF1 in TGF-β Signaling
(A and B) Physical association between Smad and δEF1. SMC lysates were incubated with the indicated antibodies (IP) and immunoprecipitated. δEF1 (A) or Smad3 (B) in the immunoprecipitate was detected with the corresponding antibodies.
(C and D) Effects of SRF, Smad3, and δEF1 on the SM α-actin promoter. NIH3T3 cells were cotransfected with full-length SM α-actin luciferase plasmid and expression vectors encoding SRF, Smad3, and/or δEF1, with or without constitutively active ALK5 (caALK5), as indicated. The luciferase activity was normalized to that of the SM α-actin reporter cotransfected with empty plasmid. Bars indicate relative luciferase activity and SE.
(E) Induction of δEF1 protein by TGF-β stimulation. Rat aortic SMCs were cultured in serum-free defined medium for 4 days, after which TGF-β (2.5 ng/ml) was added to the medium. Whole-cell lysates were subjected to Western analyses of δEF1 and β-tubulin.
(F) Cells transfected with either δEF1-siRNA or control siRNA and incubated in serum-free medium for 24 hr were harvested, after which their whole-cell lysates were subjected to Western analyses.
(G) Effects of δEF1 knockdown on SM α-actin expression. Either δEF1-specific siRNA or control scrambled siRNA was transfected into SMCs. Twenty-four hours later, the medium was changed to serum-free defined medium for an additional 24 hr, after which TGF-β (2.5 ng/ml) was added for the indicated times. mRNA expression was analyzed by real-time PCR. Solid lines represent gene expression in cells transfected with the scrambled siRNA; dotted lines, expression in cells transfected with δEF1-siRNA. Error bars represent SE from triplicate culture wells; ∗p < 0.05 (two-way ANOVA).
(H–J) ChIP analysis of δEF1, SRF, and Smad3 binding to the endogenous SM α-actin promoter region containing the THR under TGF-β1 stimulation. SMCs were cultured in defined serum-free medium for 4 days and then treated with TGF-β1 (2.5 ng/ml) for 12 hr. Chromatin samples prepared from these cells were subjected to ChIP analysis. PCR was carried out to detect the SM α-actin promoter. Lanes 1 and 2 show amplification of total input DNA. Lanes 3 and 4 show PCR amplification of control samples precipitated with no antibody. Lanes 5 and 6 show amplification of target sequences within the immunoprecipitates. The sequence of GAPDH, which does not contain a δEF1 binding sequence, was also amplified to confirm the specificity of the assay (H).
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6
Effects of Haploinsufficiency of δEF1 on Vascular Lesion Formation
(A–G) Real-time PCR analysis of the expression of δEF1, SIP1, and SMC markers in the aortas of wild-type, δEF1+/−, and δEF1−/− embryos. Aortas were collected from embryos of each genotype (n = 3, 4, and 3, respectively), after which total RNA was isolated from the collected samples, and real-time PCR was carried out. The relative numbers of transcripts were normalized to those of 18S, after which expression of the transcript for each genotype was further normalized to that of the wild-type embryo. Error bars represent SE in real-time PCR data.
(H) Representative photomicrographs of transverse sections of femoral arteries harvested from wild-type and δEF1+/− mice 4 weeks after injury with a catheter guide wire. The sections were stained with hematoxylin-eosin.
(I) Neointimal/medial wall area ratios; bars are means ± SE; n = 4 for each genotype.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7
Effects of Adenoviral Overexpression of δEF1 on Vascular Injury Responses
(A) Representative photomicrographs of transverse sections of rat common carotid arteries harvested 2 weeks after balloon injury and injection of empty adenoviral vector (Aa, Ac, and Ae) or Ad-δEF1 (Ab, Ad, and Af). (Aa and Ab) The sections were stained with hematoxylin-eosin. (Ac and Ad) Immunohistochemical staining of δEF1 protein (red). (Ae and Af) Immunohistochemical staining of SM α-actin protein (blue). Nuclei were counterstained with nuclear fast red. Arrows indicate the internal elastic laminae. Scale bars represent 100 μm.
(B) Neointimal/medial wall area ratios; bars are means ± SE; n = 8 for each group.
(C–J) Real-time PCR analysis of mRNA expression. Solid lines represent mRNA expression in arteries infected with empty adenovirus; dotted lines, expression in arteries infected with Ad-δEF1; n = 3 in each group. Copy number of each transcript was normalized to that of 18S, after which the expression was further normalized to that in arteries without balloon injury and adenovirus injection. Graphs indicate relative copy number and SE; ∗p < 0.05 (two-way ANOVA).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions