Journal: Biomedicines

Article Title: Celastrol and Triptolide Suppress Stemness in Triple Negative Breast Cancer: Notch as a Therapeutic Target for Stem Cells

doi: 10.3390/biomedicines9050482

Figure Lengend Snippet: List of antibodies and dilution factor used for flow cytometry and western blot.

Article Snippet: Rabbit anti-Notch4 , Cell signalling , 2423S , - , 1-1000.

Techniques: Flow Cytometry, Western Blot

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Notch4 (A-12) , Santa Cruz , sc-393893;.

Techniques: Virus, Control, Expressing, Plasmid Preparation, Recombinant, Isolation, MTT Assay, Western Blot, Software

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , RT-PCR of Notch1 - 4 transcripts in lung T reg and T eff cells isolated from PBS, OVA and OVA+UFP mouse groups (n=5). b , c , Flow cytometric analysis, cell frequencies and mean fluorescence intensity (MFI) of Notch4 expression on lung T reg and T eff cells in the respective treated groups (n=15). d , Flow cytometric analysis and cell frequencies of Notch4 expression on OT-II + CD4 + Foxp3 + T cells generated in co-cultures with sham or OVA 323–339 +UFP-pulsed alveolar macrophages without or with IL-6 or anti-IL-6R mAb (n=5). e , Flow cytometric analysis and cell frequencies of Notch4 + Helios – and Helios + lung T reg cells isolated from the respective treated groups (n=5). f , Flow cytometric analysis and cell frequencies of Notch4 expression on in vitro differentiated T reg cells derived from naive CD4 + T cells isolated from Foxp3 YFPCre , Foxp3 YFPCre Il6r Δ/Δ and Foxp3 YFPCre Stat3 Δ/Δ mice and either untreated or treated with IL-6 (n=6). g , ChIP assays for the binding of STAT3 and control (IgG) antibodies to the Notch4 promoter in lung T reg cells of OVA+UFP-treated Foxp3 YFPCre , and Foxp3 YFPCre Stat3 Δ/Δ mice (n=6). Each symbol represents one mouse. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis ( b,c,f ); two-way ANOVA with Sidak’s post hoc analysis ( a,d,e,g ). *P<0.05, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Fluorescence, Expressing, Generated, In Vitro, Derivative Assay, Binding Assay, Control

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a - c , Flow cytometric analysis, cell frequencies and (MFI) of Notch1, 2 and 3 expression on lung T reg and T eff cells in Foxp3 YFPCre (n=5). d , Cell frequencies of Notch4 expression on OT-II + CD4 + Foxp3 + T cells generated in co-cultures with sham or OVA 323–339 +UFP-pulsed alveolar macrophages without or with IL-1β, IL-25, IL-33, TSLP or TNF (n=5). e , ChIP assays for the binding of STAT3 and control (IgG) antibodies to the Notch1, 2 and 3 promoters in lung T reg cells of OVA+UFP-treated Foxp3 YFPCre , and Foxp3 YFPCre Stat3 Δ/Δ mice (n=5). Each symbol represents one mouse. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis ( a-c ); two-way ANOVA with Sidak’s post hoc analysis ( d,e ). **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Expressing, Generated, Binding Assay, Control

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , RT-PCR analysis of Notch4 expression in CD4 Cre mice in B-cells and T-cells (n=5). b , RT-PCR analysis of Notch4 expression in Foxp3 YFPCre mice in both T reg and T eff cells (n=5). c , d , IL-4 and IFN-γ expression in lung Foxp3 + CD4 + T reg . ( c ) and Foxp3 – CD4 + T eff cells. ( d ) derived from the respectively treated Foxp3 YFPCre , CD4 Cre Notch 4 Δ/Δ and Foxp3 YFPCre Notch 4 Δ/Δ mice (n=5). e , Airway hyperresponsiveness in Foxp3 YFPCre sensitized either with PBS or OVA, then challenged with OVA+UFP following transfer of OTII + Foxp3 YFPCre or OTII + Foxp3 YFPCre Notch4 Δ/Δ iT reg cells (n=5). f , Eosinophil numbers for the respective mouse groups (n=5). g , IL-4, IL-13, IL-17 and IFNγ expression in lung Foxp3 – CD4 – T eff cells. Each symbol represents one mouse (n=5). Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis ( a,c,d ); One-way ANOVA with Dunnett’s post hoc analysis ( e,f ). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , Representative PAS-stained sections of lung tissues isolated from Foxp3 YFPCre , CD4 Cre Notch4 Δ/Δ or Foxp3 YFPCre Notch4 Δ/Δ mice segregated into PBS, OVA or OVA+UFP-treated groups (200X magnification). b , Inflammation scores in the respective lung tissues. c , AHR in the respective mouse groups in response to methacholine. ( d , e ) serum total and OVA-specific IgE concentrations. f , g , absolute numbers of lung CD4 + T cells and eosinophils. ( h , i ) IL-13 and IL-17 expression in lung Foxp3 + CD4 + T reg ( h) , and Foxp3 – CD4 + T eff cells. ( i ). Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis ( b - i ). **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments. (White bars n=15), (black bars n=5) and (grey bars n=15).

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Staining, Isolation, Expressing

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , scheme of the house dust mite (HDM) airway inflammation protocol. b , Representative PAS-stained sections of lung tissues isolated from Foxp3 YFPCre or Foxp3 YFPCre Notch4 Δ/Δ mice segregated into PBS, OVA or OVA+UFP-treated groups (200X magnification). c , Inflammation scores in the respective lung tissues. d , AHR in the respective mouse groups in response to methacholine. e , serum total IgE concentrations. ( f - h ), absolute numbers of lung CD4 + T cells, neutrophils and eosinophils. i , k , IL-4, IL-13, IL-17 and IFNγ expression in lung Foxp3 + CD4 + T reg ( i ) and Foxp3 – CD4 + T eff cells ( k ). Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis (c - k ). **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments. n=5 mice per group.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Staining, Isolation, Expressing

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , Scheme for the chronic airway inflammation mouse protocol b , Representative Sirius-Red-stained sections of lung tissues isolated from Foxp3 YFPCre or Foxp3 YFPCre Notch4 Δ/Δ mice segregated into PBS, OVA or OVA+UFP-treated groups (200X magnification). c , Collagen disposition measurement in the respective lung tissues. d , AHR in the respective mouse groups in response to methacholine. e , f , absolute numbers of lung CD4 + T cells and eosinophils. g , h , IL-4, IL-13, and IL-17 expression in lung Foxp3 + CD4 + T reg ( g ) and Foxp3 – CD4 + T eff cells ( h ). i , Serum OVA-specific IgE titers in the respective groups. Each symbol represents an independent sample. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis ( c-h). *P<0.05, ****P<0.0001. Data representative of two or three independent experiments. n=5 mice per group.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Staining, Isolation, Expressing

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , Volcano plot of differential gene expression in Foxp3 YFPCre versus Foxp3 YFPCre Notch4 Δ/Δ T reg cells treated with OVA+UFP. FDR, false discovery rate; log2FC, log2(fold change). b , Enrichment pathway analysis of Hippo and Wnt pathways. c, Flow cytometric analysis and cell frequencies of Yap1 expression on lung T reg cells in the respective treated groups (n=5). d, Flow cytometric analysis and cell frequencies of β-Catenin expression on lung T reg cells in the respective treated groups (n=5). e, representative histogram, cell frequencies and MFI of phospho-Mob1 expression on lung T reg cells in the respective treated groups (light grey bar n= 4, dark grey bar n=4, light red bar n=5 and dark red bar n=4). f, representative histogram, cell frequencies and MFI of phospho-Lats1 T1079 expression on lung T reg cells in the respective treated groups (light grey bar n= 4, dark grey bar n=4, light red bar n=5 and dark red bar n=4). Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis ( c,d ); one-way ANOVA with Dunnett’s post hoc analysis ( e,f ). ***P<0.001, ****P<0.0001.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Gene Expression, Expressing

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , flow cytometric analysis and frequencies of IL13 + ILC2 (Lineage – T1/ST2 + cells) in mice of respective genotypes treated as indicated (n=5). b , c , In vitro suppression assays using ILC2 from OVA+UFP-treated Foxp3 YFPCre mice and lung T reg cells of the respective genotypes, treated as indicated (n=4) . d , GDF15 transcripts in T reg cells of Foxp3 YFPCre , Foxp3 YFPCre Notch4 Δ/Δ and Foxp3 YFPCre Ctnnb1 Δ/Δ (n=5). e , flow cytometric analysis and frequencies of GDF15 + lung T reg cells in the respective mouse genotypes treated as indicated (n=5). f , flow cytometric analysis and frequencies of IL-13 induced in naive ILC2 stimulated with IL-33, GDF15 or both (n=3). g , IL-13 expression in naive ILC2 incubated with Notch4 hi T reg cells from OVA+UFP treated mice without or with blocking GDF15 peptide (n=6). h , In vitro suppression assays using lung T reg cells and ILC2 isolated from OVA+UFP-treated Foxp3 YFPCre mice and incubated without or with GDF15 blocking peptide (n=4). Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis ( a - e , h ); One-way ANOVA with Dunnett’s post hoc analysis ( f,g ). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: In Vitro, Expressing, Incubation, Blocking Assay, Isolation

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , d , Representative PAS-stained sections of lung tissues isolated from Foxp3 YFPCre and Foxp3 YFPCre Notch4 Δ/Δ with either PBS or OVA+UFP, the latter either alone or supplemented with GDF15 or GDF15 blocking peptide, as indicated (200X magnification), Inflammation score for the respective mouse groups (n=10). b , e , AHR in Foxp3 YFPCre and Foxp3 YFPCre Notch4 Δ/Δ treated as indicated (n=10). c , f , Frequencies and absolute numbers of ILC2, eosinophils, IL-4, and IL-13, expression in lung Foxp3 – CD4 + T eff cells in the respective groups (n=10) g, AHR in Rora Cre and Rora Cre Il4/Il13 Δ/Δ treated as indicated (n=5). h, Frequencies and absolute numbers of eosinophils, ILC2, IL-4, and IL-13, expression in lung Foxp3 – CD4 + T eff cells in the respective groups (n=5). Error bars indicate SEM. Statistical tests. One-way ANOVA with Dunnett’s post hoc analysis. ( a,c,d,f ), two-way ANOVA with Sidak’s post hoc analysis ( b , e , g , h ); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Staining, Isolation, Blocking Assay, Expressing

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , b , Flow cytometric analysis, cell frequencies and MFI of Notch4 expression on circulating T reg cells ( a ) and T eff cells ( b ) of control and asthmatic subjects, the latter segregated for asthma severity (control: n=39; mild n=31; moderate: n=27; severe: n=11). c , flow cytometric analysis, cell frequencies and MFI of Notch4 expression on Helios + versus Helios – circulating T reg cells of control and asthmatic subjects (control: n=13; mild n=9, moderate n=14; severe: n=11). d , e , Flow cytometric analysis, cell frequencies and MFI of Yap ( d ) and β-catenin ( e ) expression on circulating T reg cells of control and severe asthmatic subjects (control n=24; mild n=15; moderate n=15; severe: n=11). f , Serum GDF15 concentrations in moderate and severe asthmatic subjects plotted as a function of Notch4 expression on circulating T reg cells (n=21). g , In vitro suppression third party CD4 + T cells (T eff ) by the Notch4 hi versus Notch4 lo T reg cells from severe asthmatics compared to T reg cells of control subjects (n=2 subjects, 3 replicates per dilution per subject). h , In vitro suppression assays of ILC2 activation using circulating Notch4 hi T reg cells of asthmatics subjects and control T reg cells of healthy controls, incubated at the indicated T reg cell:ILC2 ratios without or with GDF15 blocking peptide (n=5). i , Flow cytometric analysis of Notch4 expression in T reg cells of a healthy control and a severe asthmatic before and after treatment with anti-IL-6R mAb (n=1). Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis ( a - e ); simple linear regression analysis ( f ); two-way ANOVA with Sidak’s post hoc analysis ( g,h ); ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Expressing, Control, In Vitro, Activation Assay, Incubation, Blocking Assay

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , b , Flow cytometric analysis, cell frequencies and mean fluorescence intensity (MFI) of Notch1, 2 and 3 expression in peripheral blood T reg cells ( a ) and T eff cells ( b ) of control and asthmatic subjects, the latter segregated for asthma severity (control n=22, M.P n= 15, Mod n= 16. S.P n=11). c , Flow cytometric analysis and cell frequencies of Notch4 peripheral blood T reg cells of healthy control, food allergy (FA), eczema and FA+eczema (Control n=37, FA n= 28, Eczema n=10 and FA+Eczema n=20) d , Serum GDF15 concentrations in asthmatic subjects plotted as a function of Notch4 expression on circulating T reg cells (n=73) e , Cell frequencies of Notch4 expression in peripheral blood T reg cells in healthy subjects, allergic and non-allergic asthmatics (control = 56, non-allergic n=21, allergic n=85). Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis. ( a - c,e ); simple regression analysis ( d ). ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: T reg cells at these different concentrations, 1:5, 1:10 or 1:20, from Foxp3 YFPCre mice either sham treated or treated OVA+UFP with high Notch4 expression with or without the addition of anti-Notch4 mAb (clone: HMN4–14, Bioxcell) in concentration of 10ng/ml, T reg cells from Foxp3 YFPCre Notch4 Δ/Δ , Foxp3 YFPCre Ctnnb1 Δ/Δ or Foxp3 YFPCre Yap1 Δ/Δ Wwtr1 Δ/Δ mice treated with OVA+UFP.

Techniques: Fluorescence, Expressing, Control

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Primers of genes for real-time quantitative polymerase chain reaction

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: Marker

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch signals regulate SG-originated cell stemness and are implicated in embryonic SG morphogenesis in vivo . Schematic drawings of DAPT injection and checking points, i.e. E12.5, E17.5, P1, P5 and P14 ( a ). Immunohistochemistry staining of Notch4 ( b) , Oct4 ( c) and Krt19 ( d) . Immunofluorescence staining of Notch4 and Krt14 ( e ). Red arrows (b): Notch4-expressing positions. Black arrows (b–d) and white arrows (e): secretory coils of SG. Red dotted box (b–e): two critical time points during SG morphogenesis (i.e. E17.5 and P14). Scale bar (b): 200 μm, 50 μm; (c–e): 50 μm. DAPT N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, E embryonic day, Krt keratin, P postpartum day, SG sweat gland

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vivo, Injection, Immunohistochemistry, Staining, Immunofluorescence, Expressing

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 specifically targets Krt19-positive epidermal stem cells and Krt14-positive SG progenitor cells in vivo . Schematic drawings of anti-Notch4 monoclonal antibodies injection and checking points, i.e. E17.5 and P14 ( a ). Immunohistochemistry staining of Notch4 ( b ), Oct4 ( c ) and Krt19 ( d ). Immunofluorescence staining of Notch4 (red color) and Krt14 (green color) ( e ). Flow chart of this study ( f ). Red arrows (b): Notch4-expressing positions. Scale bar (b): 200 μm, 50 μm; (e): 50 μm. DAPT N -[ N -(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, E embryonic day, GO Gene Ontology, iSG induced sweat gland, Krt keratin, MSCs mesenchymal stem cells, P postpartum day, SG sweat gland, VPA valproic acid

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vivo, Bioprocessing, Injection, Immunohistochemistry, Staining, Immunofluorescence, Expressing

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 is one of the niche-dual-responding genes and is related to MSC stemness in the 3D matrix in vitro . Pairwise comparisons of ( a ) ‘3D MSC vs 2D MSC’, ( b ) ‘3D iSG vs 2D MSC’ and ( c ) ‘3D iSG vs 3D MSC’ revealed differentially expressed genes. ( d ) GO analysis showed these differentially expressed genes were enriched in cell stemness- (green font) and angiogenesis- (yellow font) related GO terms. Overlapping filtered out 76 genes and the heat map shows their transcriptional profiles. The dotted box indicates a cluster of structural cue-responsive genes. ( e ) The intersection of these 76 genes with ‘GO:0035239 tube morphogenesis’-enriched genes resulted in six candidate genes, i.e. Hmox1, Notch4, Cited1, Pdgfb, E2f8 and Mylk. GO Gene Ontology, iSG induced sweat gland, MAPK mitogen-activated protein kinase, MSCs mesenchymal stem cells

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vitro

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 specifically regulated Oct4-related stemness of MSCs in 3D matrix in vitro . ( a ) Inhibiting Notch signaling slowed down the proliferation rate of MSCs. Immunofluorescence staining of Notch4 and Oct4 in 2D culture system ( b ) and 3D culture system ( c ). ( d ) Western blotting images of Notch4 and Oct4 in response to administration of DAPT or VPA. ( e ) Relative density quantifications of Notch4 and Oct4 protein levels; n = 3. Gene expression of Notch4 and Oct4 exhibit a similar profile in both 2D culture system ( f ) (n = 3) and 3D culture system ( g ) (n = 3). Gene expressions of Notch1, 2, 3 and cell stemness markers (Nanog and Sox2) in 2D culture system ( h ) (n = 3) and 3D culture system ( i ) (n = 3). Green dotted lines indicate gene expression levels of ‘2D MSC’ group (f–i). Scale bar (a): 200 μm; (b, c): 50 μm. DAPT N -[ N -(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, DMSO dimethyl sulfoxide, KDa kilodalton, MSCs mesenchymal stem cells, NS no significant difference, RT-qPCR real-time quantitative polymerase chain reaction, VPA valproic acid

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vitro, Immunofluorescence, Staining, Western Blot, Gene Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 mediates SG differentiation of MSCs in the 3D model in vitro . Immunofluorescence staining of Notch4 (red color) and Krt18 (green color) in 2D ( a ) and 3D ( b ) culture system. Western blotting of Notch4 and Krt18 in 2D and 3D culture system ( c ). Gene expression of Notch4 ( d ), Krt18 ( e ), Krt14 ( f ) and ATP1a1 ( g ); n = 3. Scale bar (a): 50 μm; (b): 20 μm. ATP1a1 ATPase Na+/K+ transporting subunit alpha 1, DAPT N -[ N -(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, DMSO dimethyl sulfoxide, KDa kilodalton, Krt keratin, MSCs mesenchymal stem cells, NS no significant difference, PBS phosphate-buffered saline, RT-qPCR real-time quantitative polymerase chain reaction, VPA valproic acid

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vitro, Immunofluorescence, Staining, Western Blot, Gene Expression, Saline, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Signaling activated by nucleolar localised Notch4 Intracellular Domain underlies protection from genomic damage

doi: 10.1101/670588

Figure Lengend Snippet: Notch4 signaling protects from apoptosis triggered by genotoxic agents. A , Induction of apoptotic nuclear damage in MDA-MB-231 cells pretreated with siRNA to Notch4 or a scrambled control for 24 h and then continued untreated (UT) or treated with etoposide (10μM) or 5-FU (10μM) for another 24 h in serum free medium. The inset shows percent Notch4 mRNA levels in scrambled and Notch4 siRNA transfected cells. B , Induction of apoptotic nuclear damage in MDA-MB-231 cells expressing the indicated plasmids and treated with etoposide or 5-FU for 48 h as in A. C , Representative confocal images of MDA-MB-231 cells stained for endogenous Notch4 (green) and Nucleolin (red) as described in methods (Manders Correlation Coefficient: 0.73 ± 0.23). D , Representative confocal images of MDA-MB-231 cells stained with the antibody to Notch4 in cells pre-treated with vehicle control or GSI-X (10μM) for 24 h in serum free medium. E-F , Percent apoptotic nuclear damage in HEK cells expressing GFP or NIC4-GFP and treated with etoposide or 5-FU (E) or 4NQO (5μM) (F) and cultured for 48 h in serum free medium. G , Representative confocal images of HEK cells co-expressing NIC4-GFP and Fibrillarin-RFP imaged 24h after transfection (Manders Correlation Coefficient: 0.87 ± 0.15). H , Representative confocal images of MDA-MB-231 cells co-expressing NIC4-GFP and Fibrillarin-RFP imaged 24 h after transfection (Manders Correlation Coefficient: 0.96 ± 0.07). In A, B, E and F, cells were stained with Hoechst 33342 to visualize nuclei. Data represent the mean ± S.D. of three independent experiments. Scale bar: 5μm

Article Snippet: Antibodies to Notch4 (2423) and Nucleolin (14574) and anti-rabbit Alexa 543 were from Cell Signaling Technology (MA, USA).

Techniques: Control, Transfection, Expressing, Staining, Cell Culture

Journal: bioRxiv

Article Title: Signaling activated by nucleolar localised Notch4 Intracellular Domain underlies protection from genomic damage

doi: 10.1101/670588

Figure Lengend Snippet: Notch4 localisation in breast cancer cell lines. A , Representative confocal images of indicated cell lines stained for endogenous Notch4 (green) or Nucleolin (red) as described in methods. Scale bar: 5μm B , Co-localisation of Nucleolin (red) with Notch4 (green) in indicated cell lines quantified by Manders Coefficient as described in methods and shown as mean ± S.D. C , Representative confocal images of some of the cell lines in A , stained for endogenous Notch1 (mN1A, green) and counterstained with Hoechst 33342 as described in methods. Scale bar: 5μm D , Induction of apoptotic nuclear damage in Hs578T cells pretreated with siRNA to Notch4 or Notch1 or scrambled control for 24 h and then cultured untreated (UT) or treated with etoposide (10μM) for another 24 h in serum free medium. Inset shows percent mRNA levels in scrambled control and siRNA transfected cells. E , mRNA levels of indicated genes in MDA-MB-231 cells treated with siRNA to Notch4 or Notch1 or NCL or scrambled control for 48 h. Data show the mean ± S.D. of two independent experiments in D and three independent experiments in E. ns: not significant, p > 0.05 (Student’s t-test).

Article Snippet: Antibodies to Notch4 (2423) and Nucleolin (14574) and anti-rabbit Alexa 543 were from Cell Signaling Technology (MA, USA).

Techniques: Staining, Control, Cell Culture, Transfection

Journal: bioRxiv

Article Title: Signaling activated by nucleolar localised Notch4 Intracellular Domain underlies protection from genomic damage

doi: 10.1101/670588

Figure Lengend Snippet: Nucleolar localization of NIC4 controls anti-apoptotic activity. A , Schematic of the putative NoLS sequence in NIC4; NIC4-3RA, with three arginine residues replaced by alanine to perturb the NoLS; and the additional NoLS at the NIC4 N-terminal; inset: Representative confocal images of HEK cells expressing NIC4-3RA GFP (left) and NoLS NIC4 GFP (right). Scale bar: 5μm. B-C , Induction of apoptotic nuclear damage in cells expressing GFP, NIC4-GFP or NIC43RA-GFP, treated with etoposide (10μM) (B) or 5-FU (10μM) (C) for 48h in serum free medium. D , Percent apoptotic nuclear damage in cells expressing GFP, NIC4-GFP or NoLS-NIC4 GFP, treated with etoposide for 48 h in serum free medium. E , Relative transcript levels of Hes1, Hes5 and 47S rRNA in cells transfected with indicated plasmids and cultured for 36 h in complete medium. F , Relative transcript levels of indicated genes in cells transfected with GFP, NIC4-GFP or NIC43RA-GFP and cultured for 36 h in complete medium following treatment with siRNA to RBPj-κ or NCL or scrambled control. Inset shows percent RBPj-κ mRNA levels in scrambled and siRNA transfected cells. Data plotted are mean ± S.D. of three independent experiments. G , The schematic (not drawn to scale) summarizes key observations of the current study. We show that NIC4 localizes to the nucleolus and demonstrate dependency on nucleolar proteins – Nucleolin and Fibrillarin – for protection from genotoxic agents. Further, NIC4 mediated anti-apoptotic activity is dependent on canonical regulators of the DNA damage response. The experiments reveal a functional role for a Nucleolar localization sequence (NoLS) in NIC4, which agrees with NIC4 regulation of pre-ribosomal RNA transcription reported in this study.

Article Snippet: Antibodies to Notch4 (2423) and Nucleolin (14574) and anti-rabbit Alexa 543 were from Cell Signaling Technology (MA, USA).

Techniques: Activity Assay, Sequencing, Expressing, Transfection, Cell Culture, Control, Functional Assay

Journal: Nature Communications

Article Title: NOTCH4 ΔL12_16 sensitizes lung adenocarcinomas to EGFR-TKIs through transcriptional down-regulation of HES1

doi: 10.1038/s41467-023-38833-7

Figure Lengend Snippet: a ChIP analysis of NOTCH4 binding to the HES1 promoter in 293 T cells and luciferase analysis of NOTCH4 to HES1. ( n = 3 biologically independent experiments). b The predicted binding sites of STAT3 and NICD4 to HES1 promoter. c ChIP analysis of NOTCH4 and p-STAT3 binding to the HES1 promoter in 293 T cells. ( n = 3 biologically independent experiments). d Luciferase analysis of NOTCH4 and STAT3 to HES1 in 293 T cells ( n = 3 biologically independent experiments). e Western blot analysis in PC-9GR/PC-9OR. f and g ChIP analysis of NOTCH4 and p-STAT3 binding to the HES1 promoter in PC-9GR/PC-9OR cells treated with siNOTCH4 or siSTAT3. ( n = 3 biologically independent experiments). h and j The tumor growth curve of CDX models with siNOTCH4, siSTAT3 or their combination treatments. ( n = 5 CDXs). ( i and k ) The tumor weight of CDX models with siNOTCH4, siSTAT3 or their combination treatment. ( n = 5 CDXs). l and n The tumor growth curve of PDX models of PC-9GR/PC-9OR with siNOTCH4, siSTAT3 or their combination treatments. ( n = 5 PDXs). m and o The tumor weight of PDX models of PC-9GR/PC-9OR with siNOTCH4, siSTAT3 or their combination treatments. ( n = 5 PDXs) mean ± SD. An unpaired two-sided student t-test was performed in Fig. 6a–d, and 6f–o. Three biologically independent experiments were performed for Fig. 6e. Source data are provided as a Source Data file.

Article Snippet: The membranes were blocked in 5% skim milk for 2 hours and incubated overnight with the diluted (1:1000) primary antibodies at 4°C: NOTCH4 full length (Abcam, ab166605, 1:500), NOTCH4 (NICD4) (CST, #2423), HES1 (CST, #11988), PI3K (CST, #4292), Akt (CST, #4691), phospho-Akt (Ser473) (CST, #4060), MAPK (CST, #9102), P21 (CST, #2947), HEY1 (CST, #5315), phospho-STAT3(Tyr705) (CST, #9145), STAT3 (CST, #9139), NF-κB (CST, #8242), JAK1 (CST, #3332) and GAPDH (CST, #5174).

Techniques: Binding Assay, Luciferase, Western Blot

Journal: Oncotarget

Article Title: DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through notch hyper-activation

doi:

Figure Lengend Snippet: A. q(RT) PCR mRNA levels of Notch targets following DMXL2 depletion B. Representative immunofluorescence images of Hey2 following siDMXL2 showing Hey2 nuclear signal was impaired. Bars represent 400 uM C. Representative western blot showing reported TM Notch accumulates following DMXL2 depletion. Antibodies were used as in 1A D. Representative blot showing Notch3 and 4ICD reduced chromatin accumulation following siDMXL2. Antibodies were used as in 2C. Knock down was performed for 48 or 72 hrs (for mRNA or protein extraction respectively). (*) P < 0.05 (**) P < 0.01 (***) P < 0.001. Every experiment is an average of three independent experiments. Bars represent standard deviation.

Article Snippet: Antibodies against Notch1 and Notch3 were purchased from Cell Signalling (3608, and 2889) and Notch4 was purchased from Santa Cruz (Sc-5594 H-225).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Knockdown, Protein Extraction, Standard Deviation