Journal: Stem Cell Research & Therapy

Article Title: Activation of Notch1 signalling promotes multi-lineage differentiation of c-Kit POS /NKX2.5 POS bone marrow stem cells: implication in stem cell translational medicine

doi: 10.1186/s13287-015-0085-2

Figure Lengend Snippet: Forced NICD expression activation of Notch1 signalling in total and c-Kit POS /NKX2.5 POS bone marrow mesenchymal stem cells. Total bone marrow mesenchymal stem cells (BMSCs) and c-Kit POS /NKX2.5 POS BMSCs were infected with NICD-Ad or NC-Ad, with uninfected cells serving as an additional control (MOCK). After 8 days, the cells were harvested for (A) immunofluorescence staining for notch receptor intracellular domain (NICD) and Hes1, and (B) real-time quantitative RT-PCR analysis of Hes1 mRNA expression. For immunofluorescence staining, target proteins were detected with fluorescein isothiocyanate-conjugated IgG. (C) Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured by confocal microscopy and merged. Scale bar: 50 μm. Triplicate experiments were performed for real-time quantitative RT-PCR analysis ( n = 3). *** P <0.001 versus other groups. NC-Ad, negative control-expressing adenovirus; NICD-Ad, NICD-expressing adenovirus.

Article Snippet: NICD cDNA (5,468 to 7,820 nucleotides; NM_001105721) was obtained from the cDNA library of Genechem (Shanghai, China) with the following primers: 5′-AGG TCG ACT CTA GAG GAT CCC GCC ACC ATG CGC AAG CGC AGG CGG CA-3′ and 5′-TCC TTG TAG TCC ATA CCC TTA AAT GCC TCT GGA ATG TGG G-3′.

Techniques: Expressing, Activation Assay, Infection, Control, Immunofluorescence, Staining, Quantitative RT-PCR, Confocal Microscopy, Negative Control

Journal: Stem Cell Research & Therapy

Article Title: Activation of Notch1 signalling promotes multi-lineage differentiation of c-Kit POS /NKX2.5 POS bone marrow stem cells: implication in stem cell translational medicine

doi: 10.1186/s13287-015-0085-2

Figure Lengend Snippet: Exogenous Jagged1 activation of Notch1 signalling in total and c-Kit POS /NKX2.5 POS bone marrow mesenchymal stem cells. Total bone marrow mesenchymal stem cells (BMSCs) and c-Kit POS /NKX2.5 POS BMSCs were classified into Jagged1, IgG, N -(2S-(3,5-difluorophenyl)acetyl)- l -alanyl-2-phenyl-1,1-dimethylethyl ester-glycine (DAPT), dimethyl sulfoxide (DMSO), and MOCK treatment groups (see ). After 8 days, activation of Notch1 signalling was assessed by immunofluorescence staining of notch receptor intracellular domain (NICD) and Hes1 protein expression (A) and quantitative RT-PCR analysis of Hes1 mRNA expression (B) . For immunofluorescence staining, target proteins were detected with fluorescein isothiocyanate-conjugated IgG. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. Representative images were captured by confocal microscopy. Triplicate experiments were performed for real-time quantitative RT-PCR analysis ( n = 3). *** P <0.001 versus other groups.

Article Snippet: NICD cDNA (5,468 to 7,820 nucleotides; NM_001105721) was obtained from the cDNA library of Genechem (Shanghai, China) with the following primers: 5′-AGG TCG ACT CTA GAG GAT CCC GCC ACC ATG CGC AAG CGC AGG CGG CA-3′ and 5′-TCC TTG TAG TCC ATA CCC TTA AAT GCC TCT GGA ATG TGG G-3′.

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Confocal Microscopy

Journal: Cancer Science

Article Title: KIAA 0247 inhibits growth, migration, invasion of non‐small‐cell lung cancer through regulating the Notch pathway

doi: 10.1111/cas.13539

Figure Lengend Snippet: Antibodies used for western blotting in the present study

Article Snippet: NICD , Wanlei Bio , wl03097a , Rabbit , 1:1000.

Techniques: Western Blot

Journal: Frontiers in Immunology

Article Title: Inhibition of the intracellular domain of Notch1 results in vascular endothelial cell dysfunction in sepsis

doi: 10.3389/fimmu.2023.1134556

Figure Lengend Snippet: Overexpression of NICD reversed LPS-caused endothelial dysfunction. (A, B) The expression of NICD in Ea.hy 926 cells was tested by Western blot after transfected with blank plasmids, vector or NICD plasmids. Representative images (A) and three experiments replicates (B) were displayed. (C) CCK8 assay was used to evaluate the proliferation of Ea.hy 926 cells treated with the indicated conditions. (D) The apoptosis of Ea.hy 926 cells treated with indicated conditions was tested with Annexin V-FITC/PI staining by flow cytometry. Representative images (D) -left) and statistical analysis of apoptosis (D) -right) for Ea.hy 926 cells transfected by blank plasmids, vector or NICD plasmids after LPS treatment were shown. (E) The endothelial permeability was determined by the transwell assay after being treated with the indicated conditions. (F) The migration of Ea.hy 926 cells was tested by wounding healing assay. Representative images (F) -left) and statistical analysis of migration (F) -right) for Ea.hy 926 cells transfected by blank plasmids, vector or NICD plasmids after LPS treatment were shown. (G, H) The protein levels of cleaved PARP, NICD, Hes1, VE-Cad, Zo-1, PTEN, and p-AKT was tested in LPS-treated Ea.hy 926 cells with or without NICD-transfected. Representative images (G) and three experiments replicates (H) were displayed. The data were obtained from three independent experiments. Data are shown as the mean ± SEM. * p < 0.05.

Article Snippet: The expression plasmid containing NICD sequence (aa 1757-2555) was purchased from Genechem (China) and transfected into Ea.hy 926 cells using Lipofectamine 3000 reagents (Invitrogen, USA).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, CCK-8 Assay, Staining, Flow Cytometry, Permeability, Transwell Assay, Migration

Journal: Frontiers in Immunology

Article Title: Inhibition of the intracellular domain of Notch1 results in vascular endothelial cell dysfunction in sepsis

doi: 10.3389/fimmu.2023.1134556

Figure Lengend Snippet: The ubiquitination of NICD was regulated by USP8. (A) Ubiquitinated NICD measured by immunoprecipitation with anti-NICD antibody and immunoblotting with anti-ubiquitin antibody in LPS treated Ea.hy 926 cells with or without melatonin. (B) The mRNA expression of a series of DUBs in Ea.hy 926 cells was tested by real-time qPCR. (C) The protein expression of USP8 and USP11 was detected by western blot in LPS-treated Ea.hy 926 cells with or without melatonin. (D) The interaction of NICD and USP8 in Ea.hy 926 cells was detected using Co-immunoprecipitation. (E) The expression of NICD and USP8 in Ea.hy 926 cells transfected with different USP8 siRNA. Scramble siRNA was used as negative control (si-NC). (F, G) The stability of NICD was tested in Ea.hy 926 cells transfected without or with si-USP8 at the indicated time after CHX (20 μM) treatment (F) . The quantitative analysis of (G) with Image J. (H, I) The ubiquitination of NICD in Ea.hy 926 cells with indicated treatment through NICD immunoprecipitation (H) and input (I) . Data are shown as the mean ± SEM. * p < 0.05. ns, no significance.

Article Snippet: The expression plasmid containing NICD sequence (aa 1757-2555) was purchased from Genechem (China) and transfected into Ea.hy 926 cells using Lipofectamine 3000 reagents (Invitrogen, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Transfection, Negative Control