Sorting of SMCs from RA-induced SM22α^−/−

<p>^{<b><i>LacZ</i></b>}<b> ESCs.</b> (A) Morphology change of SM22α^−/−LacZ ESCs under the treatment of DMSO (upper panel) or 10⁻⁵ M RA (lower panel) respectively as indicated time. (B) Comparison of β-gal staining of cells treated with either DMSO (left panel) or 10⁻⁵ M RA (right panel). The upper panel showed that β-gal staining positive cells accumulated with RA treatment time, while only sporadic β-gal staining positive cells existed in DMSO-treated cell population. The lower panel showed representative magnified images by day 8. Scale bar = 100 µm. (C–D) Using 5-chloromethylfluorescein di-β-D-galactopyranoside (FDG) to react with intracellular glutathione. In <i>LacZ</i>-positive cells derived from RA-induced SM22α^−/−LacZ ESCs, the FDG–glutathione adduct was converted to a bright green fluorescent product and <i>LacZ</i>-positive cells were subsequently sorted through a GFP channel (C) and cultured (D). Left panel: bright field; middle panel: GFP channel; right panel: merge. Scale bar = 100 µm. (E) Immunofluorescence staining of SMCs derived from sorted <i>LacZ</i>-positive cells with SMC-specific marker α-SMA antibody. The nuclei were co-stained with DAPI. Scale bar = 100 µm. (F) Gene expression of sorted cells analyzed by quantitative real-time PCR, compared to undifferentiated ESCs (control, Ctrl) or cells before sorting. Before: before sorting; After: after sorting. *<i>p</i><0.05.</p

Representative SEM micrographs of NF scaffold and sorted SMCs seeded on the scaffold.

<p>(A–B) Low and high magnification images of the NF scaffold respectively. The cells were seeded and cultured for 24 hours and observed at low (C) or high (D) magnifications. Arrows indicate the cell aggregates inside the pores of scaffolds.</p

Histology of constructs implanted subcutaneously for 2 weeks.

<p>(A) β-gal staining. (Left) blank scaffold implants; (Right) cell-scaffold construct implants. Blue: positive <i>LacZ</i> staining; Red: nuclei. (B) H&E staining of cell-scaffold construct implants. Scale bar = 200 µm.</p

Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3

<div><p>Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells.</p> </div

Effect of Diapin on blood glucose levels in diabetic mice.

<p>After oral loading of Diapin (Δ, 0.5 mg/g; ▪, 1 mg/g) by gavage, OGTT was performed in male (<b>A</b>) <i>ob/ob</i> (n = 10), (<b>B</b>) <i>db/db</i> (n = 10) diabetic mice, (<b>C</b>) <i>KKay</i> (n = 9), and (<b>D</b>) the high fat diet-induced obesity mice (n = 10), in which the wild type male C57BL/6J mice were fed with high fat diet for ten weeks. *<i>p</i><0.05, compared with the controls.</p

Egr-1 binding sites are essential for Rad promoter activation.

<p>(A) Nucleotide sequence between base pairs -76 and -51bp of Rad promoter. Egr-1 binding sites are boxed. The mutated bases are indicated in boldface. (B) Wild-type and mutant forms of pRad-155 were cotransfected with pcDNA3.1-Egr-1* or pcDNA3.1 into 293A cells and luciferase activity were determined. The fold induction of Lucifearse activity by Egr-1 was shown in the lower panel; (C) RASMCs were transfected with pRad-155 promoter reporter or its mutant forms then treated with PDGF and luciferase activity were determined (n = 4, **<i>p</i><0.01). (D) pRad-3050 and pRad-3050m were transfected into RASMCs then treated with PDGF and luciferase activity were determined (n = 4, **<i>p</i><0.01).</p

Effect of Diapin on blood glucose levels in C57BL/6J mice during OGTT.

<p><b>A.</b> Effect of Diapin on blood glucose. The mice were fasted overnight and orally loaded with glucose (2 mg/g, bw) in the control (n = 10) and glucose (2 mg/g) plus Diapin (4 µmol/g, equal to 1 mg/g) in the treated group (n = 10). <b>B.</b> Effect of the amino acids of Diapin on blood glucose. In the treated group (n = 10), the mice were loaded with glucose and mixture of amino acids of Diapin (G, G, L; 4 µmol/g for each amino acid). <b>C.</b> Effect of the dipeptides on blood glucose. In the treated groups, the mice were loaded with glucose and dipeptides (GG or GL, 4 µmol/g). <b>D.</b> Effect of a control peptide on blood glucose. In the treated group (n = 10), the mice were loaded with glucose and a tripeptide (4 µmol/g). Blood glucose levels were measured every 30 min after glucose loading. *<i>P</i><0.05, compared with the controls.</p

Effect of Diapin on non-fasting blood glucose levels in <i>KKay</i> diabetic mice.

<p>(A) Male <i>KKay</i> diabetic mice and (<b>B</b>) adult male C57BL/6J mice under non-fasting condition were given either vehicle in control groups or Diapin (1 mg/g bw, n = 9/group) in the treated groups. After Diapin loading, the blood glucose levels were measured every 30 min. *<i>P</i><0.05, compared with the controls.</p

Time dependent effect of Diapin on blood glucose level in <i>KKay</i> diabetic mice.

<p>Male <i>KKay</i> diabetic mice were randomly divided into control and treated groups. In control group (•), the mice were fed with regular chaw diet. The mice in the treated groups were fed with regular chow diet containing 6 (Δ) and 12 g/kg Diapin (▪), respectively. The casual blood glucose levels were monitored weekly. n = 10, *<i>p</i><0.05, compared with the control.</p

Interactions between wild-type and R90C and C49Y mutants of NOTCH3.

<p>(A–C) NOTCH3-NOTCH3 self-association is mediated by the extracellular domain. 293 cells were transiently co-transfected with cDNAs expressing the NOTCH3 extracellular domain tagged with HA (A, B, and C were transfected with WT, R90C, and C49Y, respectively) and cotransfected with myc tagged NOTCH3 ectodomain constructs as shown. The proteins were extracted after 40∼48 hours and immunoprecipitated with HA monoclonal antibody. Co-immunoprecipitates of cells cotransfected with plasmid combinations shown were analyzed by immunoblotting (IB). Quantification of protein bands from four independent experiments failed to show a significant difference in IP complexes with any of the NOTCH3 combinations. (D) Effect of divalent cations on stability of NOTCH3 homophilic interactions and the stability of NOTCH3 homophilic complexes. Coimmunoprecipitation was performed using the HA antibody on lysates of cells transfected with plasmid combinations shown on the right column. Immunoprecipitates were washed extensively with buffers containing calcium or magnesium, and then analyzed by western blotting. All of the HA and myc tagged proteins run on SDS gels at an apparent molecular weight of 175 kDa. We verified the finding of numerous groups that common epitope tags do not mediate protein interaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#pone.0044964-Zhang2" target="_blank">[64]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#pone.0044964-Pulipparacharuvil1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#pone.0044964-Nazarenko1" target="_blank">[66]</a> (not shown). All experiments were performed more than 6 times.</p