murine nicd1  (Addgene inc)

Journal: Scientific Reports

Article Title: Class II phosphatidylinositol 3-kinase-C2α is essential for Notch signaling by regulating the endocytosis of γ-secretase in endothelial cells

doi: 10.1038/s41598-021-84548-4

Figure Lengend Snippet: PI3K-C2α is required for Dll4-induced Notch signaling in HUVECs. ( a ) Representative Western blot images showing the effects of PI3K-C2α knockdown on Dll4-induced NICD1 production. PI3K-C2α-siRNA- or control (ctrl)-siRNA-transfected HUVECs were incubated on Dll4- or BSA-coated plates and 24 h later underwent Western blot analysis using anti-NICD1 and anti-PI3K-C2α antibodies. Right two panels show the quantified data of PI3K-C2α and NICD1 protein levels (n = 3). Statistical significance was assessed with two-tailed unpaired Student’s t test for PI3K-C2α and two-way ANOVA followed by Bonfferroni’s post-hoc test for NICD1. ( b ) The effects of PI3K-C2α knockdown on Dll4-induced Notch target gene expression. PI3K-C2α-siRNA- or ctrl-siRNA-transfected HUVECs were incubated on Dll4- or BSA-coated plates and 24 h later subjected to qPCR analysis (n = 5). Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. ( c ) Relative mRNA expression levels of Notch paralogues compared with NOTCH1 . The values on the top of bars represent the means ± SEM. HUVECs were cultured on BSA-coated plates for 24 h and underwent qPCR analysis (n = 3). ( d ) The effects of PI3K-C2α knockdown on mRNA expression of Notch paralogues. PI3K-C2α-siRNA- or ctrl-siRNA-transfected HUVECs were incubated on Dll4- or BSA-coated plates and 24 h later subjected to qPCR analysis (n = 3). In a-d, data are presented as means ± SEM from three to five independent experiments. Statistical significance was presented as * P < 0.05, ** P < 0.01 or *** P < 0.001, respectively. ns: not significant.

Article Snippet: To overexpress NICD1, HUVECs were transfected with 3xFlag-tagged murine NICD1 expression plasmids (Addgene plasmid #20183, 2–4 μg) using Amaxa HUVEC Nucleofector Kit.

Techniques: Western Blot, Knockdown, Control, Transfection, Incubation, Two Tailed Test, Targeted Gene Expression, Expressing, Cell Culture

Journal: Scientific Reports

Article Title: Class II phosphatidylinositol 3-kinase-C2α is essential for Notch signaling by regulating the endocytosis of γ-secretase in endothelial cells

doi: 10.1038/s41598-021-84548-4

Figure Lengend Snippet: HEY1, HEY2 and NOTCH3 are Notch1 target genes in HUVECs. ( a ) The effects of Notch1 knockdown on Dll4-induced NICD1 production in HUVECs. Notch1-siRNA- or ctrl-siRNA-transfected cells were incubated on Dll4- or BSA-coated plates and 24 h later subjected to Western blot analysis for Notch1 and NICD1. β-actin was analyzed as a loading control. Right two panels show the quantified data of Notch1 (TM-IC + NEXT) and NICD1 protein levels (n = 3). Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. TM-IC: transmembrane and intracellular domain of full-length Notch1. NEXT: Notch1 extracellular truncation. ( b ) The effects of Notch1 knockdown on Dll4-induced Notch target gene expression. Notch1-siRNA- or ctrl-siRNA-transfected HUVECs were incubated on Dll4- or BSA-coated plates and 24 h later underwent qPCR analysis (n = 3). Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. ( c ) Expression of 3xFLAG-tagged-NICD1 in HUVECs. Cells were transfected with the indicated expression vectors or not, and 24 h later harvested for Western blot analysis using anti-FLAG, anti-Notch1, and anti-β-actin antibodies. GFP denotes transfection with the GFP expression plasmid (4 μg) as a control. ( d ) The effects of NICD1 overexpression on Notch target gene expression in HUVECs. Cells were transfected with FLAG-tagged-NICD1 expression plasmid (4 μg) or GFP expression plasmid (4 μg) or mock-transfected, and 24 h later harvested for qPCR analysis (n = 3). Statistical significance between mock-treated cells and NICD1-overexpressing cells was assessed with one-way ANOVA followed by Bonfferroni’s post-hoc test. Statistical significance was presented as * P < 0.05, ** P < 0.01 or *** P < 0.001, respectively. ns: not significant.

Article Snippet: To overexpress NICD1, HUVECs were transfected with 3xFlag-tagged murine NICD1 expression plasmids (Addgene plasmid #20183, 2–4 μg) using Amaxa HUVEC Nucleofector Kit.

Techniques: Knockdown, Transfection, Incubation, Western Blot, Control, Targeted Gene Expression, Expressing, Plasmid Preparation, Over Expression

Journal: Scientific Reports

Article Title: Class II phosphatidylinositol 3-kinase-C2α is essential for Notch signaling by regulating the endocytosis of γ-secretase in endothelial cells

doi: 10.1038/s41598-021-84548-4

Figure Lengend Snippet: PI3K-C2α is required for Dll4-induced Notch signaling in endothelial cells but not in smooth muscle cells. ( a , d ) Relative mRNA expression level of Notch paralogues compared with NOTCH1 . The values on the top of bars represent the means ± SEM. Isolated total RNAs of HMVECs-L ( a ) and HAoSMCs ( d ) were subjected to qPCR analysis, respectively. (n = 3). ( b , e ) The effects of PI3K-C2α knockdown on Dll4-induced NICD1 production in HMVECs-L ( b ) and HAoSMCs ( e ). PI3K-C2α-siRNA- or ctrl-siRNA-transfected cells were incubated on Dll4- or BSA-coated plates for 24 h. Then cell lysate was subjected to Western blot analysis for NICD1, PI3K-C2α, and β-actin. The right panels show the quantified data of NICD1 protein level (n = 3). Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. ( c , f ) The effects of PI3K-C2α knockdown on Dll4-induced Notch target gene expression in HMVECs-L ( c ) and HAoSMCs ( f ). PI3K-C2α-siRNA- or ctrl-siRNA-transfected cells were incubated on Dll4- or BSA-coated plates, and 24 h later harvested for qPCR analysis (n = 3–4). Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. Statistical significance was presented as * P < 0.05, ** P < 0.01 or *** P < 0.001, respectively. ns: not significant.

Article Snippet: To overexpress NICD1, HUVECs were transfected with 3xFlag-tagged murine NICD1 expression plasmids (Addgene plasmid #20183, 2–4 μg) using Amaxa HUVEC Nucleofector Kit.

Techniques: Expressing, Isolation, Knockdown, Transfection, Incubation, Western Blot, Targeted Gene Expression

Journal: Scientific Reports

Article Title: Class II phosphatidylinositol 3-kinase-C2α is essential for Notch signaling by regulating the endocytosis of γ-secretase in endothelial cells

doi: 10.1038/s41598-021-84548-4

Figure Lengend Snippet: PI3K-C2α and clathrin are required for γ-secretase complex-mediated cleavage of truncated Notch1. ( a ) Representative Western blot images showing the effects of PI3K-C2α knockdown on the abundance of cell surface Notch1 and NEXT in HUVECs. PI3K-C2α-siRNA- or ctrl-siRNA-transfected cells were incubated on Dll4- or BSA-coated plates, and 24 h later total cell lysate and biotin-labeled surface proteins were prepared, followed by Western blot analysis for Notch1 and β-actin. Right panels show the quantified data of cell surface TM-IC level (n = 4) and the abundance of NEXT in total cell lysate as NEXT over NEXT + TM-IC (n = 5), respectively. Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. ( b ) Representative confocal images of double immunofluorescent staining with anti-Notch1 and either anti-LAMP1 (left) or anti-EEA1 (right) antibodies. Cells received γ-secretase inhibitor DAPT (1 μM) before seeding and were incubated on Dll4-coated plates for 12 h before the fixation. Nuclei were stained with DAPI (asterisks). DAPT treatment almost completely abolished Dll4-induced nuclear translocation of Notch1. Note the endolysosomal accumulation of Notch1 (arrowheads) was observed in DAPT-treated cells. Scale bar, 20 μm. ( c , d ) The effects of CHC knockdown on Dll4-induced NICD1 production ( c ) and Notch target gene expression ( d ). CHC-siRNA- or ctrl-siRNA-transfected HUVECs were incubated on Dll4- or BSA-coated plates and 24 h later subjected to Western blot analysis (n = 5) and qPCR analysis (n = 3–4), respectively. Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. ( e ) The effects of CHC knockdown on cell surface Notch1 level and NEXT abundance in HUVECs. CHC-siRNA- or ctrl-siRNA-transfected HUVECs were incubated on Dll4- or BSA-coated plates for 24 h. Total cell lysate and biotin-labelled surface proteins were prepared and analyzed as in ( a ). Right panels show the quantified data of cell surface TM-IC level and the abundance of NEXT in total cell lysate (n = 3). Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test. Statistical significance was presented as * P < 0.05, ** P < 0.01 or *** P < 0.001, respectively. ns: not significant.

Article Snippet: To overexpress NICD1, HUVECs were transfected with 3xFlag-tagged murine NICD1 expression plasmids (Addgene plasmid #20183, 2–4 μg) using Amaxa HUVEC Nucleofector Kit.

Techniques: Western Blot, Knockdown, Transfection, Incubation, Labeling, Staining, Translocation Assay, Targeted Gene Expression

Journal: Scientific Reports

Article Title: Class II phosphatidylinositol 3-kinase-C2α is essential for Notch signaling by regulating the endocytosis of γ-secretase in endothelial cells

doi: 10.1038/s41598-021-84548-4

Figure Lengend Snippet: Schematic summary showing the role of PI3K-C2α in Dll4-Notch1 signaling in ECs. After the processing by Furin-like convertases (S1 cleavage) in the Golgi, cleaved Notch receptors, which are now heterodimers composed of the extracellular domain and TM-IC, are delivered to the plasma membrane. γ-secretase complexes, which are hetero-tetramers composed of Presenilin, Nct, PEN-2 and Aph-1, exist at the plasma membrane and are constitutively internalized in a manner dependent on clathrin and PI3K-C2α. Dll4 binding induces Notch1 cleavage at the juxtamembrane site (S2 cleavage) by ADAM to generate NEXT. NEXT is internalized in a manner independent of clathrin and PI3K-C2α. Internalized NEXT is cleaved at the transmembrane domain (S3 cleavage) by γ-secretase at the endolysosomal compartment to release NICD1. NICD1 translocates to the nucleus and forms the transcriptional activator complex to stimulate the expression of Notch target genes including HEY1 , HEY2 and NOTCH3 . EE: early endosomes, LE: late endosomes, LY: lysosomes.

Article Snippet: To overexpress NICD1, HUVECs were transfected with 3xFlag-tagged murine NICD1 expression plasmids (Addgene plasmid #20183, 2–4 μg) using Amaxa HUVEC Nucleofector Kit.

Techniques: Clinical Proteomics, Membrane, Binding Assay, Expressing