Journal: Journal of Cardiovascular Development and Disease

Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

doi: 10.3390/jcdd6040039

Figure Lengend Snippet: Primers qPCR, ChIP and EMSA.

Article Snippet: Polyclonal anti-Nos3 antibody was purchased from Microm (Rabbit, 1:50) and used on OCT embedded and cryo-sectioned fixed tissue.

Techniques: Sequencing

Journal: Journal of Cardiovascular Development and Disease

Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

doi: 10.3390/jcdd6040039

Figure Lengend Snippet: Modulation of gene expression in Krox20−/− embryos. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Krox20−/− , Tie2-cre;Krox20f/f , and Wnt1-cre;Krox20f/f mice. ( B , C ) Cross-sectional H&E images through the aortic valve of Krox20+/+ ( B ) and Krox20−/− ( C ) littermate embryos. At E18.5, left and right-coronary leaflets are observed in BAV of Krox20−/− ( C ) embryos. ( D ) Real-time qPCR analyses were performed from isolated aortic valve of Krox20+/+ ( n = 5) and Krox20−/− ( n = 5) embryos at E18.5. qPCR showing normal levels of Alk2 , Gata5 , and Hey1 , and altered expression of Nos3 , Notch1 , and Hey2 in the aortic valve of Krox20−/− embryos at E18.5. qRT-PCR experiments were performed in triplicate and expressed as mean ±SEM (* p < 0.05 using Mann–Whitney test). Scale bars: 100 μm.

Article Snippet: Polyclonal anti-Nos3 antibody was purchased from Microm (Rabbit, 1:50) and used on OCT embedded and cryo-sectioned fixed tissue.

Techniques: Gene Expression, Isolation, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of Cardiovascular Development and Disease

Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

doi: 10.3390/jcdd6040039

Figure Lengend Snippet: Abnormal aortic valve morphology in Krox20+/−;Nos3+/− mice. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Nos3+/− , Krox20+/− , Nos3+/− ; Krox20+/− and Nos3−/− mice. ( B – D ) H&E images showing representative Nos3+/− and Krox20+/− with tri-leaflets aortic valve, and an example of Nos3+/− ; Krox20+/− aortic valve with 2 leaflets. Aortic valve leaflets of Nos3+/− ; Krox20+/− mice appear equal in size. ( E ) Real-time qPCR demonstrates a reduction of Nos3 at a transcriptional level in Nos3+/− ; Krox20+/− and Krox20−/− compared to wild-type embryos ( n = 5 for each genotype). qPCR experiments were performed in triplicate and expressed as mean ± SEM (* p < 0.05 using Mann–Whitney test). Scale bars: 100 μm.

Article Snippet: Polyclonal anti-Nos3 antibody was purchased from Microm (Rabbit, 1:50) and used on OCT embedded and cryo-sectioned fixed tissue.

Techniques: MANN-WHITNEY

Journal: Journal of Cardiovascular Development and Disease

Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

doi: 10.3390/jcdd6040039

Figure Lengend Snippet: Endothelial nitric oxide synthase (Nos3) expression is altered in Krox20 deficient mice. ( A – F ) Immunohistochemistry showing Nos3 protein (green) in the aortic valve of wild-type (WT, A ), Nos3−/− ( B ), Krox20−/− ( C ), Nos3+/− ( D ), Krox20+/− ( E ), and Nos3+/− ; Krox20+/− ( F ) embryos at E18.5. Immunohistochemistry showing abundant expression of Nos3 in the valve endothelial cells (arrows). Note the reduction of Nos3 expression in the mesenchyme of Krox20−/− ( C ) aortic valve leaflets (arrowhead; compared C with A ). Scale bars: 100 μm.

Article Snippet: Polyclonal anti-Nos3 antibody was purchased from Microm (Rabbit, 1:50) and used on OCT embedded and cryo-sectioned fixed tissue.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Cardiovascular Development and Disease

Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

doi: 10.3390/jcdd6040039

Figure Lengend Snippet: Krox20 promotes the transcriptional activity of the Nos3 proximal promoter. ( A ) Schematic representation of the 1.5-kb Nos3 proximal promoters. The putative Krox20 binding sites located around −513 bp and −136 bp on Nos3 promoters are represented by black squares. Asterisk (*) indicates conservation of Krox20-binding sites with human and rat sequences. Numbers indicate position of the sequences from the ATG. ( B ) EMSA showing binding of Krox20 to the wild-type K1 (GTGTGGGAC) and K2 (GTGTGGGTT) motifs. Mutation of the K1 and K2 motif impairs Krox20 binding. ( C ) Relative luciferase activity in Cos7 cells transiently co-transfected with reporter constructs containing a 1544 bp and 265 bp regions of Nos3 promoter cloned upstream of the luciferase gene, 10 ng of control pGL4.74( hRluc /TK) vector and 50, 150, and 300 ng of CMV-Krox20 or CMV control expressing vectors. Transfection of CMV- Krox20 has a trans-activating effect on −1544 bp reporter. Mutation of the K2 motif abolishes the trans-activation of Krox20 on the −1544 bp reporter. Data is represented as a fold change in luciferase activity normalized to Renilla . ( D ) qPCR showing significant enrichment of DNA/Krox20 complexes on Krox20-binding sites within the Nos3 (regions A) proximal promoters following chromatin immunoprecipitation using dissected outflow tract (OFT) and left ventricle (LV) from E13.5 hearts. Anti-Histone H3 was used as positive control of immunoprecipitation. Primers used for qPCR correspond to Nos3 proximal promoter region ( Nos3-A , Nos3-B ) as indicated in ( A ). Primers in region without Krox20 binding sites (no binding site) were used as a negative control. Relative quantities of each chromatin bound fragment were normalized relative to the amount of input DNA. Note significant enrichment in region A within the Nos3 promoters. ( E ) Nos3 transcriptional level was quantified by qRT-PCR in rat AVICs, 24 h, 48 h, and 72 h after Krox20 transfection. Experiments were performed in triplicate and expressed as means ± SEM.

Article Snippet: Polyclonal anti-Nos3 antibody was purchased from Microm (Rabbit, 1:50) and used on OCT embedded and cryo-sectioned fixed tissue.

Techniques: Activity Assay, Binding Assay, Mutagenesis, Luciferase, Transfection, Construct, Clone Assay, Control, Plasmid Preparation, Expressing, Activation Assay, Chromatin Immunoprecipitation, Positive Control, Immunoprecipitation, Negative Control, Quantitative RT-PCR

Journal: Journal of Cardiovascular Development and Disease

Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

doi: 10.3390/jcdd6040039

Figure Lengend Snippet: Absence of Nos3 does not affect Krox20 contribution. ( A , B ) Embryos were harvested from Krox20 Cre/+ ;Rosa tdTomato mice at E18.5 and immunohistochemistry is performed to detect Tomato expression (red) as an indicator of recombination, while Pecam (green) identifies endothelial cells in the aortic valve. No major difference is observed between Nos3−/− and control littermate embryos. ( C – F ) X-gal staining was performed on −31/−23.5 Krox20/LacZ transgenic mice to follow the Krox20 -expressing cells at E9.5 ( C , D ), and E13.5 ( E,F ) stages. ( C , D ) At E9.5, β-galactosidase (β-gal) activity is detected in migratory neural crest cells. No defect is observed in Nos3−/− compared with control embryos (compared D with C ). ( E , F ) Transverse section through the outflow tract cushions at E13.5 showing β-gal-positive cells in the arterial valve leaflets. AoV, aortic valve; ba1, branchial arch 1; PV, pulmonary valve; NCC, neural crest cells. Scale bars: 100 μm ( A , B ); 50 μm ( C – F ).

Article Snippet: Polyclonal anti-Nos3 antibody was purchased from Microm (Rabbit, 1:50) and used on OCT embedded and cryo-sectioned fixed tissue.

Techniques: Immunohistochemistry, Expressing, Control, Staining, Transgenic Assay, Activity Assay