recombinant human notch3 ecd  (R&D Systems)

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Schematic representation of Notch signaling. (1) Furin (S1 cleavage) cleaves the NOTCH3 precursor protein in the Golgi system, resulting in a non‐covalently bound bipartite protein that is transported to the cell surface. (2) A mechanical traction force is applied to the NOTCH3 ECD when a Notch ligand binds to the EGF repeats 10–11, exposing the extracellular NRR near the cell membrane, which consists of LNR and the heterodimerization domain (in green). Subsequently, ADAM17 cleaves the C‐terminal portion of the heterodimerization domain (S2‐cleavage). (3) The NEXT, which is made up of a RAM domain, the ANK domains, a PEST domain, and a transmembrane domain, is cleaved by the γ‐secretase (S3‐cleavage) releasing the N3ICD. (4) The N3ICD binds to the CSL protein and together with the co‐activator Mastermind‐like (MAM) trigger downstream gene transcription in the nucleus. (5) The NOTCH3 ECD and ligand are normally endocytosed by the ligand‐expressing cell and degraded in the lysosome. Schematic representation of NOTCH3 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) mutations. NOTCH3 ECD contains 34 EGF repeat domains, each of which has six cysteine residues (WT). Mutations in CADASIL change the number of cysteines to an uneven number of cysteines (Mutant). These unpaired cysteines residues result in incorrect EGF repeat folding, irregular protein folding which leads to an enhanced NOTCH3 ECD multimerization. Distribution of the cysteine‐altering mutations that cause CADASIL are shown. In the CADASIL mutant NOTCH3 ECD, the endocytosis is hampered, and NOTCH ECD remains outside of the VSMC and starts to accumulate and aggregate around the vessels. ADAM17, a disintegrin and metalloproteinase domain‐containing protein 17; ANK, ankyrin repeats; EGF, epidermal growth factor; HD, heterodimerization domain; LNR, Lin‐Notch repeats; PEST, proline (P), glutamic acid (E), serine (S), and threonine (T) degradation domain; RAM, Rbp‐associated molecule domain; TM, transmembrane domain.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Membrane, Expressing, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Schematic representation of NOTCH3 and NOTCH3 EGF 1–5 . NOTCH3 represents the full‐length protein, and NOTCH3 EGF 1–5 represents the NOTCH3 protein with exon 1–5 fused with a myc‐His‐Tag at the C‐terminus used for purification of the aggregated protein. Western blot of the NOTCH3 EGF 1–5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α‐myc antibody. Western blot of NOTCH3 EGF 1–5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF 1–5 WT and R133C were visualized on a western blot using an α‐myc antibody. The purified proteins and the aggregates were visualized after 1–5 days incubation by western blot using α‐myc antibody under non‐reducing conditions. Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Purification, Western Blot, Incubation

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Schematic pipeline of the subcutaneous active immunization in the TgN3R182C150 mouse model. Antibody titer validation of serum from TgN3R182C150 CADASIL mice immunized with NOTCH3 EGF 1–5 aggregates (vaccinated, n = 11) and PBS (sham, n = 9) at 4, 5, and 7 months old. A direct ELISA with NOTCH3 aggregate‐coated plates and different dilutions of serum was performed. Error bars indicate standard error of the mean (SEM).

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Biomarker Discovery, Direct ELISA

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐immunized mice at 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (7 and 18 months), sham and NOTCH3 EGF 1–5 ‐immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α‐SMA antibody (green). Scale bar = 20 μm. Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF 1–5 ‐immunized ( n = 10), sham ( n = 8), and non‐vaccinated ( n = 6) TgN3R182C150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months ( n = 3) of age versus NOTCH3 EGF 1–5 ‐immunized ( n = 10), sham ( n = 8), and non‐vaccinated ( n = 6) TgN3R182C150 mice at 7 months of age. Statistical significance was assessed using a Brown‐Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons (NOTCH3 ECD deposits (% of vessel area): 7 m.o. vs. Sham, ns P = 0.9424; 7 m.o. vs. Vaccinated, ns P = 0.9809; 7 m.o. vs. 18 m.o. ** P = 0.0046; Sham vs. Vaccinated, ns P = 0.9999; Sham vs. 18 m.o. ** P = 0.0032; Vaccinated vs. 18 m.o. ** P = 0.0032. NOTCH3 ECD deposits (number/1,000 μm 2 ): 7 m.o. vs. Sham, ns P = 0.7877; 7 m.o. vs. Vaccinated, ns P = 0.8358; 7 m.o. vs. 18 m.o. ** P = 0.0069; Sham vs. Vaccinated, ns P = 0.9995; Sham vs. 18 m.o. ** P = 0.0044; Vaccinated vs. 18 m.o. * P = 0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P = 0.1169; 7 m.o. vs. Vaccinated, ns P > 0.9999; 7 m.o. vs. 18 m.o. *** P = 0.0009; Sham vs. Vaccinated, ns P = 0.0577; Sham vs. 18 m.o. ** P = 0.007; Vaccinated vs. 18 m.o. **** P < 0.0001, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: The target genes NOTCH3 , Hes1 , Hey1 and Nrip2 on TgN3R182C150 mice at 5 and 12 months of age. Three biological replicates were run in three technical replicates. Error bars indicate standard deviation (SD).

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF 1–5 ‐immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti‐perlecan antibody (green). Scale bar = 20 μm. Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3‐ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non‐vaccinated 3‐month‐old TgN3R182C150 ( n = 3) and 7‐month‐old TgN3R182C150 ( n = 6) mice and 18‐month‐old TgN3R182C150 ( n = 3). Quantification of NOTCH3‐ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3‐ECD stained area and average size per vessel revealed a significant decrease in NOTCH3‐ECD deposition in brain arteries and capillaries between NOTCH3 EGF 1–5 ‐immunized ( n = 11), sham ( n = 8), and non‐vaccinated TgN3R182C150 ( n = 6) mice. Statistical significance was assessed using a Brown‐Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons (NOTCH3 ECD deposits (% of vessel area): 3 m.o. vs. 18 m.o. ** P = 0.0029; 3 m.o. vs. 7 m.o. *** P = 0.0004; 7 m.o. vs. Sham, ns P = 0.7326; 7 m.o. vs. Vaccinated, ** P = 0.003; Sham vs. Vaccinated, **** P < 0.0001. NOTCH3 ECD deposits (number/1,000 μm 2 ): 3 m.o. vs. 18 m.o. ** P = 0.0038; 3 m.o. vs. 7 m.o. *** P = 0.0002; 7 m.o. vs. Sham, ns P = 0.9913; 7 m.o. vs. Vaccinated, * P = 0.021; Sham vs. Vaccinated, *** P = 0.001. NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. * P = 0.0215; 3 m.o. vs. 7 m.o. ** P = 0.0029; 7 m.o. vs. Sham, ns P = 0.3622; 7 m.o. vs. Vaccinated, * P = 0.0428; Sham vs. Vaccinated, ** P = 0.0038, ns = non‐significant). Dotted lines indicate quartiles and dashed thicker lines are the median. Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: NOTCH3 ECD was detected in the whole blood serum of the non‐treated TgN3R182C150 mice at 3 months ( n = 9) of age and further increased at 7 months ( n = 6) of age. Serum from Notch3 −/− and C57Bl6/J WT mice were included as negative controls. Statistical analysis was performed using unpaired Student's t ‐test with Welch's correction (3 m.o. vs. 7 m.o. *** P = 0.0005). Error bars indicate standard error of the mean (SEM). NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated ( n = 10) TgN3R182C150 mice vs. sham ( n = 9). Statistical analysis was performed using unpaired Student's t ‐test with Welch's correction (Sham vs. Vaccinated * P = 0.0196). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Serum from 7‐month‐old non‐vaccinated TgN3R182C 150 mice ( n = 4) was monitored in the absence or presence of serum (1:1 dilution) from sham‐and vaccinated C57Bl6/J WT mice. No significant changes in NOTCH3 ECD concentration was observed after dilution adjustment. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Dunnett's multiple comparisons test (TgN3R182C 150 vs. TgN3R182C 150 + C57BL6/J WT Sham, ns P = 0.8383; TgN3R182C 150 vs. TgN3R182C 150 + C57BL6/J WT Vaccinated, ns P = 0.7000; ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images show microglia stained with anti‐CD68 antibody (red) and Iba1 antibody (green). Scale bar = 20 μm. Quantification of CD68‐stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF 1–5 ‐immunized ( n = 6), sham ( n = 4), and non‐vaccinated TgN3R182C150 ( n = 5) mice. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Tukey's multiple comparisons test (% microglia with CD68 staining: 7 m.o. vs. Sham ns P = 0.7393; Sham vs. Vaccinated ** P = 0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P = 0.9131; Sham vs. Vaccinated * P = 0.0157, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Representative images of TgN3R182C150, sham‐, and NOTCH3 EGF 1–5 ‐immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar = 20 μm. Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF 1–5 ‐immunized ( n = 11), sham ( n = 8), and non‐vaccinated TgN3R182C150 ( n = 6) mice at 7 months of age. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Tukey's multiple comparisons test (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P = 0.7997; Sham vs. Vaccinated ns P = 0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P = 0.5362; Sham vs. Vaccinated ns P = 0.8059. NOTCH3 ECD deposits (number/1,000 μm 2 ): 7 m.o. vs. Sham ns P = 0.8392; Sham vs. Vaccinated ns P = 0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P = 0.6664; Sham vs. Vaccinated ns P = 0.8787, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Immunostaining for smooth muscle actin (ASMA) revealed that there were no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in WT (C57Bl6/J) versus TgN3R182C150 mice at 7 months of age. Scale bar = 50 μm. Immunostaining for smooth muscle actin (ASMA) shows no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in NOTCH3 EGF 1–5 ‐vaccinated versus sham‐vaccinated TgN3R182C150 mice. Scale bar = 50 μm. Immunostaining for smooth muscle actin (ASMA) shows an extensive loss of VSMC in the Notch3 −/− mice when compared with a WT (C57Bl6/J) at 3 months of age. Scale bar = 50 μm.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Immunostaining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: NIH3T3 cells were transfected with the control, WT NOTCH3, or NOTCH3 R182C plasmids, as well as the β‐gal and 12XCSL‐luc reporter plasmids and cultured on immobilized jagged2 (Jag2) in the presence of DMSO or DAPT ( n = 3 and two technical replicates). Statistical analysis was performed using 2‐way ANOVA followed by Tukey's multiple comparisons tests (Control: Fc + DMSO vs. Jag2 + DMSO ns P = 0.7223; Jag2 + DMSO vs. Jag2 + DAPT ns P = 0.817. N3WT: Fc + DMSO vs. Jag2 + DMSO *** P = 0.0004; Jag2 + DMSO vs. Jag2 + DAPT ** P = 0.0069. N3R182C: Fc + DMSO vs. Jag2 + DMSO * P = 0.0135; Jag2 + DMSO vs. Jag2 + DAPT * P = 0.0352. Jag2 + DMSO: Control Jag2 + DMSO vs. N3WT Jag2 + DMSO *** P = 0.0008; Control Jag2 + DMSO vs. N3R182C Jag2 + DMSO * P = 0.0314; N3WT Jag2 + DMSO vs. N3R182C Jag2 + DMSO ns P = 0.4187, ns = non‐significant). Error bars indicate standard error of the mean (SEM). RLU, relative luminescence units. Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Transfection, Control, Cell Culture

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: Histology of tumor invasive front. H&E staining. Scale bar, 100μm. B: Distribution of NOTCH3-positive fibroblastic cells. C: Distribution of α-SMA-positive fibroblastic cells. Cells stained brown in B and C represent positive cells for each antibody. Arrows in A , B and C indicate blood vessel layer, which is positive internal control of each antibody. D , E , F: Dual immunohistochemical analysis for α-SMA (red) and NOTCH3 (Green). Co-localization of α-SMA and NOTCH3 in fibroblasts was observed in cancer stroma. Scale bar, 50μm. Ca, cancer cells. Dotted lines in A - F indicate the interface of cancer nests and stroma. G: Kaplan-Meier curve for overall survival in relation to NOTCH3 expression in CAFs using 93 human OSCC cases. Log-rank test was used to calculate significance.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Control, Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: α-SMA and NOTCH3 expression in fibroblasts in the OSCC stroma.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: Clinico-pathological Significance of NOTCH3 expression in CAFs.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cultured alone (ctl; control) or cocultured with represented oral and maxillary SCC cell lines. 3 days after coculture, NHDFs were isolated from this coculture by using anti-Fibroblast microbeads for Western blot analysis. B and C: Immunofluorostainig using coculture with HO1-N-1 and NHDFs. Culture of NHDFs alone was used as a control. The bundles of NOTCH3-positive NHDFs (green) were observed around the AE1/AE3-positve HO1-N-1 cancer nests (red) ( B ). Double NOTCH3 and α-SMA-positive NHDFs were intervened between HO1-N-1 cancer nests. Ca, HO1-N-1 cancer nests. Dotted lines demonstrate the interface of HO1-N-1 cancer nests and NHDFs. ( C ). Scale bar, 100μm. The nuclei were counterstained by DAPI. D-G: NHDFs transfected with negative control siRNA (siCtrl) or siRNA for NOTCH3 (siN3) was cultured alone or cocultured with HO1-N-1 cells. 3 days after coculture, NHDFs were isolated from this coculture and subjected to western blot ( D ) and qPCR analyses to measure NOTCH3 ( E ), HEY1 ( F ), α-SMA ( G ). H: NHDFs were cultured alone (control), or directly cocultured with HO1-N-1 cells, or cocultured with transwell-separated HO1-N-1 cells. 3 days after culture, NHDFs were isolated and subjected to qPCR analysis. I: qPCR to measure each NOTCH mRNA expression in NHDFs isolated from coculture with HO1-N-1 cells. ND, not detected. * P < 0.05, ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Isolation, Western Blot, Transfection, Negative Control, Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: JAGGED1 expression in various oral cancer cell lines was analyzed by Western blot. B: The effect of NOTCH3 on cell proliferation. The proliferation of HO1-N-1 cells with or without recombinant human NOTCH3-Fc chimera treatment was monitored for 72hr.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A and B : Comparison of the microvessel density (MVD) between NOTCH3(-) CAFs and NOTCH3(+)CAFs cases. Immunofluorostaining for α-SMA (green), NOTCH3 (green) and CD34 (red) using human tongue OSCC samples. Ca, cancer nests. Dotted lines showed the interface of the cancer nests and stroma. Arrows demonstrated the CD34-positive microvessel. Scale bar, 50μm. The nuclei were counterstained by DAPI ( A ). Quantitative evaluation of MVD between these two groups ( B ). C-F : In vitro angiogenesis assay cocultured with HO1-N-1 cells, HUVECs and siRNA transfected NHDFs. Immunofluorostaining for NOTCH3 (green) and CD31 (red). Ca, HO1-N-1 cancer nests. Dotted lines indicate the margin of the HO1-N-1 cancer nests. Scale bar, 100μm. The nuclei were counterstained by DAPI ( C ). NHDFs isolated from this coculture were subjected to Western blot ( D ). Representative image of tube formation by HUVECs in each condition. Scale bar, 100μm ( E ). Quantitative evaluation of tube formation area (CD31-positive area) in each condition ( F ). siCtrl, negative control siRNA. siN3, siRNA for NOTCH3. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, In Vitro, Angiogenesis Assay, Transfection, Isolation, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cocultured with various cancer cell lines derived from non-oral lesions. 3 days after coculture, NHDFS were isolated from this coculture and subjected to Western blot analysis to detect NOTCH3 and α-SMA. B-D: In vitro angiogenesis assay cocultured with A549, HUVECs and siRNA transfected NHDFs. Representative images of tube formation ( B ) and its quantitative evaluation ( C ) in each condition. Scale bar, 100μm. NHDFs isolated from this angiogenesis assay were subjected to Western blot analysis ( D ). siCtrl, negative control siRNA. siN3, siRNA for NOTCH3. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Derivative Assay, Isolation, Western Blot, In Vitro, Angiogenesis Assay, Transfection, Negative Control

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cocultured with HO1-N-1 for 3 days. Prior to coculture, NHDFs were transfected with negative control siRNA or siRNA for NOTCH3. After 3 days, NHDFs were isolated from this coculture by using anti-fibroblast microbeads. NHDFs were subjected to real-time qPCR analyses to measure Notch3 expression. B-C: These isolated NHDFs were seeded on 24-well plates (2.5×10 5 cells/well) and cocultured with HUVECs (3.0×10 4 cells/well) for 4 days. Representative images of tube formation ( B ) and its quantitative evaluation ( C ) in each condition. Scale bar, 100μm. ctlNHDF(siCtl), negative control siRNA transfected NHDF isolated from culture alone. co-NHDF(siCtl), negative control siRNA transfected NHDF isolated from coculture with HO1-N-1. co-NHDF(siN3), siRNA for NOTCH3 transfected NHDF isolated from coculutre with HO1-N-1. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Negative Control, Isolation, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits

doi: 10.1186/s40478-014-0096-8

Figure Lengend Snippet: Overview of autopsy cases

Article Snippet: 1 μg recombinant human Notch3 encompassing aa 40–467 (N3EGF1-11-Fc) and IgG 1 -Fc (both R&D Systems) were dissolved in PBS and coated onto Maxisorp 96-well plates (Nunc) overnight at 4°C.

Techniques: Mutagenesis, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits

doi: 10.1186/s40478-014-0096-8

Figure Lengend Snippet: Fibronectin and fibrillin-1 accumulate in CADASIL brain vessels. Paraffin-embedded brain sections of three CADASIL patients and three healthy controls were stained for Notch3-ECD, fibronectin and fibrillin-1, counterstained with hematoxylin (blue) and analyzed by bright-field microscopy. (CAD: CADASIL patient; Ctrl: control).

Article Snippet: 1 μg recombinant human Notch3 encompassing aa 40–467 (N3EGF1-11-Fc) and IgG 1 -Fc (both R&D Systems) were dissolved in PBS and coated onto Maxisorp 96-well plates (Nunc) overnight at 4°C.

Techniques: Staining, Microscopy, Control

Journal: Acta Neuropathologica Communications

Article Title: Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits

doi: 10.1186/s40478-014-0096-8

Figure Lengend Snippet: Fibrillin-1 and Notch3-ECD do not co-localize. Frozen brain sections from three CADASIL patients were co-stained for fibrillin-1 and Notch3-ECD and analyzed by fluorescence microscopy. (CAD: CADASIL patient).

Article Snippet: 1 μg recombinant human Notch3 encompassing aa 40–467 (N3EGF1-11-Fc) and IgG 1 -Fc (both R&D Systems) were dissolved in PBS and coated onto Maxisorp 96-well plates (Nunc) overnight at 4°C.

Techniques: Staining, Fluorescence, Microscopy

Journal: Acta Neuropathologica Communications

Article Title: Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits

doi: 10.1186/s40478-014-0096-8

Figure Lengend Snippet: LTBP-1 accumulates in CADASIL brain vessels and co-localizes with Notch3-ECD aggregates. a Frozen brain sections from a CADASIL patient and a healthy control containing an arteriole (left panels) or a capillary (right panels) were stained for LTBP-1 and analyzed by fluorescence microscopy. b Frozen brain sections of three CADASIL patients were stained for LTBP-1 and Notch3-ECD and analyzed by confocal fluorescence microscopy. c LTBP-1 co-fractionates with Notch3-ECD. The final (β-ME-containing) fractions of sequentially extracted brain vessels from CADASIL patients and controls were immunoblotted for Notch3-ECD, LTBP-1 and β-tubulin. Shown is a representative blot from two independent experiments. (CAD: CADASIL patient; Ctrl: control).

Article Snippet: 1 μg recombinant human Notch3 encompassing aa 40–467 (N3EGF1-11-Fc) and IgG 1 -Fc (both R&D Systems) were dissolved in PBS and coated onto Maxisorp 96-well plates (Nunc) overnight at 4°C.

Techniques: Control, Staining, Fluorescence, Microscopy

Journal: Acta Neuropathologica Communications

Article Title: Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits

doi: 10.1186/s40478-014-0096-8

Figure Lengend Snippet: LAP accumulates in CADASIL brain vessels. a LAP co-fractionates with Notch3-ECD. The β-ME fractions of sequentially extracted brain vessels from CADASIL patients and controls were immunoblotted for LAP and β-tubulin. Shown is a representative image of two independent extractions. b LAP accumulates in the tuncia media of CADASIL patient vessels. Paraffin-embedded brain sections were stained for LAP and counterstained with hematoxylin. (CAD: CADASIL patient; Ctrl: control).

Article Snippet: 1 μg recombinant human Notch3 encompassing aa 40–467 (N3EGF1-11-Fc) and IgG 1 -Fc (both R&D Systems) were dissolved in PBS and coated onto Maxisorp 96-well plates (Nunc) overnight at 4°C.

Techniques: Staining, Control

Journal: Acta Neuropathologica Communications

Article Title: Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits

doi: 10.1186/s40478-014-0096-8

Figure Lengend Snippet: LTBP-1 binds to immobilized Notch3 in a solid-phase binding assay. a The interaction of full-length LTBP-1 derived from conditioned cell supernatants with an immobilized Notch3 fragment (N3EGF1-11-Fc) is increased 4.9-fold when compared to a control ligand (IgG-Fc). Results are expressed as mean + SEM of seven independent experiments. Bound LTBP-1 was re-evaluated by immunoblotting and predominantly detected in oligomeric form. b Schematic representation of the used LTBP-1 constructs and their domain organization including cysteine-rich repeats (orange ovals), a hybrid domain (grey ovals), EGF-like repeats (blue boxes) and V5-His or HA tags (black circles). The fibronectin, TGF-β, and fibrillin-1 binding regions are indicated. Note that LTBP-ΔC-HA was used in assays with cell supernatants and LTBP-ΔC-V5 in assays requiring purified LTBP-1. c LTBP-1 binding to Notch3 is mediated by its N-terminus. While the N-terminal deletion variant LTBP-1ΔN does not bind significantly, the C-terminal deletion variant LTBP-1ΔC, either from conditioned supernatant or in purified form, shows significant interaction. Results are expressed as mean + SEM of five (LTBP-1ΔC_V5) and four (LTBP-1ΔN_HA) independent experiments. n.s.: not significant, ***p < 0.001; **p < 0.01; Mann–Whitney Test.

Article Snippet: 1 μg recombinant human Notch3 encompassing aa 40–467 (N3EGF1-11-Fc) and IgG 1 -Fc (both R&D Systems) were dissolved in PBS and coated onto Maxisorp 96-well plates (Nunc) overnight at 4°C.

Techniques: Binding Assay, Derivative Assay, Control, Western Blot, Construct, Purification, Variant Assay, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits

doi: 10.1186/s40478-014-0096-8

Figure Lengend Snippet: LTBP-1ΔC specifically co-aggregates with mutant Notch3. a Notch3-ECD multimer formation is monitored by SIFT and data illustrated in a 2D histogram: axes represent the intensity of photons per bin in the detector channel (green channel along the x-axis, red channel along the y-axis). While monomers and homomeric multimers result in data points in the lower left histogram area and along the axes respectively, heteromeric multimers are represented as high-intensity, dual-color signals in the white sector. b Purity of proteins used in the aggregation assay. Shown are silver-stained gels containing the elution fractions after metal-ion matrix (LTBP-1ΔC or LTBP-1ΔN) or Halo-tag-mediated purification (N3EGF1-5 R183C). c SIFT data from different protein combinations: while no high-molecular-weight particles are detected with N3EGF1-5 WT, typical aggregates are formed by N3EGF1-5 R183C and when combined with LTBP-1ΔC. In all other combinations homomeric multimers are detected indicating self-aggregation. Shown are representative images of 2–5 experiments. WT: wild-type.

Article Snippet: 1 μg recombinant human Notch3 encompassing aa 40–467 (N3EGF1-11-Fc) and IgG 1 -Fc (both R&D Systems) were dissolved in PBS and coated onto Maxisorp 96-well plates (Nunc) overnight at 4°C.

Techniques: Mutagenesis, Staining, Purification, Molecular Weight