The assessment of <i>miR-1-1/133a-2</i> intragenic enhancer using the transgenic mice expressing the reporter gene, <i>LacZ</i>, and driven by the intragenic enhancer.

<p>β-Galactosidase staining (blue) was performed on embryos (<b>A</b>-<b>D</b>) and the embryos were sectioned for detailed analysis (<b>E</b>-<b>L</b>). The enhancer containing wild-type SRE directed the <i>LacZ</i> gene expression in heart and somite areas (<b>A</b>, <b>E</b> and <b>I</b>). Close-up of β-Galactosidase staining showed the cardiac-specific expression of lacZ. (<b>B</b>, <b>F</b> and <b>J</b>) The enhancer containing the mutant SRE directed the <i>LacZ</i> gene expression in somite areas but not in the heart (<b>C</b>, <b>G</b> and <b>K</b>). Close-up of β-Galactosidase staining displayed the abolishment of lacZ expression in heart (<b>D</b>, <b>H</b> and <b>L</b>). Embryos were at E10.5 (<b>A</b>-<b>H</b>) and E11.5 (<b>I</b>-<b>L</b>). HR: heart. lv: left ventricle. rv: right ventricle, la: left atrium. ra: right atrium, m: somite myotomes. White arrows indicate somite myotomes.</p

The putative SRE motif located in the intragenic enhancer of <i>miR-1-1/133a-2</i> was verified by the chromatin immunoprecipitation (ChIP) assay with sequencing assessment <i>in vivo</i>.

<p>The cell chromatins were extracted from C2C12 cells transfected with either SRF expression vector or siRNA specific for SRF, and the SRF-SRE complexes were pulled down by the anti-SRF antibody. <b>A</b>. The oligonucleotides containing the SRF-binding site were detected (left panel). The PCR product was sequenced and the sequence matched with the enhancer (right panel). <b>B</b>. Quantitative PCR showed that SRF knockdown was achieved. <b>C</b>. Quantitative ChIP assays showed that loss of SRF resulted in decreases in the SRF-SRE complexes, and non-specific PCR primers failed to detect the SRE motif. SRE-P: specific primers for SRE; NS-P: non-specific primers upstream and downstream of the SRE site.</p

Analysis of <i>miR-1-1/133a-2</i> intragenic enhancer region containing the SRE motif by luciferase assay.

<p>Empty bar represents reporter vector activity directed by the enhancer with the wild-type SRE motif. Solid bar represents reporter vector activity directed by the enhancer with the mutant SRE motif. SRF increased the luciferase reporter gene activity, and the addition of NKx2.5 facilitated SRF functional activity. Mutation of the SRF binding site resulted in a reduction of the reporter gene activity. The data in each group represents the averages of three experiments with a total of six measurements. Statistical significance was determined by unpaired, two-tailed student’s <i>t</i> test. Data are means ± S.E.</p

Effects of DAPT treatment on cell proliferation and apoptosis.

<p><b>A.</b> Immunohistochemical and immunofluorescence staining were used to determine the expression of PCNA and TUNEL in fibrosis induced by CCl₄ in liver tissues from normal, fibrosis, and DAPT-treated rats. <b>B, C.</b> Regions with positive PCNA or TUNEL staining were quantified using ImageJ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046512#s4" target="_blank">Materials and Methods</a>. The white bars represent 50 µm. ^#<i>P</i>>0.05 versus DAPT-treated rats; *<i>P</i><0.01 versus normal rats; ^##<i>P</i>>0.05 versus rats in fibrosis group; **<i>P</i><0.05 versus rats in fibrosis group.</p

DAPT treatment inhibited EMT in HSC-T6 cells.

<p>Expression of snail, vimentin, Hes1, and α-SMA in HSC-T6 cells cultured with DAPT (0.1, 0.2, 0.4, and 0.8 µmol/l) or DMSO (control) for 48 h were analyzed by Western blot analysis. The expression was normalized against β-actin. *<i>P</i><0.05 versus control group.</p

Effects of DAPT on inhibiting Notch signaling activation in fibrotic liver. A.

<p>The protein levels of Notch3-ICD, Hes1, and TGF-β1 were examined by Western blot. <b>B.</b> The expression was normalized against β-actin. *<i>P</i>>0.05 versus rats in fibrosis group. ^#<i>P</i><0.05 versus rats in fibrosis group. 1 indicates normal rats; 2 indicates rats in fibrosis group treated with DMSO and CCl₄; 3 indicates rats treated with CCl₄ and DAPT (10 mg/kg); 4 indicates rats treated with CCl₄ and DAPT (50 mg/kg).</p

The protein levels of Notch signaling components in fibrotic liver.

<p><b>A</b>. Rats treated with olive oil or CCl₄ were killed. The protein levels of Notch3-ICD, Jagged1, and Hes1 were analyzed by Western blot. <b>B.</b> The expression was normalized against β-actin. *<i>P</i><0.05 versus rats in normal group. ^#<i>P</i><0.05 versus rats at 8 weeks.</p

DAPT treatment was found to inhibit EMT in fibrotic livers in rats.

<p>Immunohistochemical staining was performed to detect the expression of vimentin, snail, E-cadherin, and TGF-β1 in livers from normal, fibrosis, and DAPT-treated rats.</p

Immunofluorescence evidence for increased Notch signaling activation in fibrotic livers.

<p>Liver sections of rats after 8 weeks of CCl₄ or olive oil treatment were stained with antibodies against Notch3, Jagged1, Hes1, and α-SMA. <b>A.</b> Notch3. <b>B.</b> Jagged1. <b>C.</b> Hes1. <b>D.</b> Liver sections of rats after 8 weeks of CCl₄ treatment were costained with antibodies against Hes1 (red) and α-SMA (green). Nuclei were stained with DAPI (blue). Images were taken by confocal fluorescent microscopy and the white bars represent 50 µm. Arrows indicate Hes1/α-SMA double positive cells.</p

Table2_Characterization of cardiac involvement in patients with LMNA splice-site mutation–related dilated cardiomyopathy and sudden cardiac death.XLSX

Introduction:LMNA splicing mutations occur in 9.1% of cases with cardiac involvement cases, but the phenotype and severity of disease they cause have not yet been systematically studied. The aim of this study was to understand the clinical and pathogenic characteristics of the LMNA splice-site mutation phenotype in patients with LMNA-related dilated cardiomyopathy (DCM) and sudden cardiac death (SCD).Methods and Results: First, we reported a novel family with LMNA-related DCM and SCD, and the clinical characteristics of all current patients with LMNA splicing mutations were further summarized through the ClinVar database. Seventeen families with a total of 134 individuals, containing a total of 15 LMNA splicing mutation sites, were enrolled. A total of 42 subjects (31.3%) had SCD. Compared without with the non-DCM group (n = 56), the patients within the DCM group (n = 78) presented a lower incidence of atrioventricular block (AVB) (p = 0.015) and a higher incidence rates of non-sustained ventricular tachycardia (p = 0.004),) and implantable cardioverter defibrillator (ICD) implantation (p = 0.005). Kaplan‒Meier survival analysis showed that the patients with pacemaker (PM) implantation had a significantly reduced the occurrence of SCD compared to patientswith those without PM implantation (log-rank p A mutation in the reported family, and pathogenic prediction analysis showed that the mutation site was extremely harmful. Next, we conducted gene expression levels and cardiac pathological biopsy studies on the proband of this family. We found that the expression of normal LMNA mRNA from the proband was significantly downregulated in peripheral blood mononuclear cells than incompared with healthy individuals. Finally, we comprehensively summarized the pathological characteristics of LMNA-related DCM, including hypertrophy, atrophy, fibrosis, white blood cell infiltration, intercalated disc remodeling, and downregulation of desmin and connexin 43 (Cx43) expression.Discussion: Above all, Cardiaccardiac involvement in patients with LMNA splice-site mutation presented with a high rate of SCD. Implanting a pacemaker significantly reduced the SCD rate in non-DCM patients with AVB. The pathogenic characterization was not only haveinvolved suppressed the expression of the healthy LMNA allele, but was also associated with abnormal expression and distribution of desmin and Cx43.</p