Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 is upregulated in LUAD tissues and cells. (A) The protein level of Notch4 in five paired LUAD tissues and adjacent non-malignant tissues. (B) The protein levels of Notch4 in A549 and H1299 cells and normal HBE cell. ( n = 8). (C) The protein levels of Notch4 in H1299 cells exposed to hypoxia for different times. ( n = 3). (D) The protein levels of Notch4 in A549 cells exposed to hypoxia for different times. ( n = 5). Data were presented as means ± SD. * p < 0.05, ** p < 0.01. LUAD, lung adenocarcinoma; HBE, human bronchial epithelial.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 gene silencing alleviated xenograft tumor growth and metastasis. (A–F) Xenograft tumor growth experiments were performed in nude mice with A549-control and A549-shNotch4 stable cells. (A) Representative images of subcutaneous tumor dissected from the nude mice were presented. (B) Representative H&E staining images in sections of tumor were presented. Magnification, ×200; Bar, 50 μm. (C) Subcutaneous tumor growth curves of the nude mice were presented. ( n = 10). (D) Subcutaneous tumor weights were presented. ( n = 10). (E) The mRNA level of Notch4 in xenograft tumor. ( n = 8–10). (F) The protein levels of Notch4, p38, p-P38, JNK, p-JNK, ERK, p-ERK in xenograft tumor. ( n = 3). (G, I) Lung metastasis experiments were performed in nude mice with A549-control and A549-shNotch4 stable cells. (G) Representative images of lung metastases were presented. (H) The numbers of visible metastatic nodules in the lungs of mice were counted. ( n = 3–4). (I) Representative H&E staining images in sections of lung tissues were presented. Magnification, ×200; Bar, 50 μm. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. H&E, hematoxylin-eosin staining. JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Control, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 is involved in the regulation of cell proliferation in A549 and H1299 cells exposed to hypoxia. (A, B) Cell viability was assessed using CCK-8 assay in A549 and H1299 cells ( n = 4–5). (C, D) A549 cell proliferation was assessed using Edu assay. ( n = 4). Magnification, ×400; Bar, 20 μm. (E, F) H1299 cell proliferation was assessed using Edu assay. ( n = 5). Magnification, ×400; Bar, 20 μm. (G) Colony formation assay was performed in A549 and H1299 cells. ( n = 3). Data were presented as means ± SD. * p < 0.05, comparison with normoxic cells treated with si-NC; # p < 0.05, comparison with hypoxic cells treated with si-NC. CCK-8, Cell Counting Kit-8; si-NC, negative control short interfering RNAs (siRNA); si-Notch4, the siRNA against Notch4.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: CCK-8 Assay, EdU Assay, Colony Assay, Comparison, Cell Counting, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 is involved in the regulation of cell migration and apoptosis in A549 and H1299 cells exposed to hypoxia. (A) Wound healing assay was performed in A549 and H1299 cells. ( n = 4). Magnification, ×40; Bar, 200 μm. (B) Cell apoptosis was assessed by Annexin-V/PI staining. ( n = 3). Analyses of apoptosis including early apoptosis (Annexin-V positive and PI negative) and late apoptosis (Annexin-V positive and PI positive) were shown. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, comparison with normoxic cells treated with si-NC; # p < 0.05, comparison with hypoxic cells treated with si-NC. si-NC, negative control short interfering RNAs (siRNA); si-Notch4, the siRNA against Notch4; PI, propidium iodide.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Migration, Wound Healing Assay, Staining, Comparison, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: ERK, JNK, and P38 MAPK signaling mediate the regulation of Notch4 overexpression on A549 and H1299 cells proliferation and apoptosis. (A) The MAPK signaling pathway were detected in A549 and H1299 cells transfected with siRNA against Notch4 or negative control siRNA. (B) Cell viability were examined in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with ERK (U0126), JNK (SP600125), or P38 (SB203580) MAPK pathway inhibitors by using CCK-8 assay. ( n = 3–4). (C) Colony formation assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580 for 24 h ( n = 4). (D) The Annexin-V/PI assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580 for 24 h ( n = 3–6). Analyses of apoptosis including early apoptosis (Annexin-V positive and PI negative) and late apoptosis (Annexin-V positive and PI positive) were shown. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CCK-8, Cell Counting Kit-8; PI, propidium iodide; si-NC, negative control short interfering RNAs (siRNA); si-Notch4, the siRNA against Notch4. Vector, negative control plasmid; Notch4, Notch4 plasmid. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Over Expression, Transfection, Negative Control, Plasmid Preparation, CCK-8 Assay, Colony Assay, Cell Counting

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: ERK, JNK, and P38 MAPK signaling mediate the regulation of Notch4 overexpression on A549 and H1299 cells migration. (A–D) Wound healing assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580. ( n = 3). Magnification, ×40; Bar, 200 μm. (E–H) Transwell migration assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580 for 24 h ( n = 3). Magnification, ×100; Bar, 50 μm. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Vector, negative control plasmid; Notch4, Notch4 plasmid. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Over Expression, Migration, Wound Healing Assay, Transfection, Plasmid Preparation, Negative Control, Transwell Migration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 interacts with ERK, JNK, and P38 in A549 cells. Endogenous co-immunoprecipitation was performed in A549 cells exposed to normoxia or hypoxia for 24 h. (A) Western blot analysis for endogenous ERK, JNK, and P38 after IP of endogenous Notch4 from A549 cells exposed to normoxia or hypoxia. (B) Western blot analysis for endogenous Notch4 after IP of endogenous ERK from A549 cells exposed to normoxia or hypoxia. (C) Western blot analysis for endogenous Notch4 after IP of endogenous JNK from A549 cells exposed to normoxia or hypoxia. (D) Western blot analysis for endogenous Notch4 after IP of endogenous P38 from A549 cells exposed to normoxia or hypoxia. (E) Whole-cell lysates were used for IB with the indicated antibodies to show expression. IP, immunoprecipitation; IB, immunoblot. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Schematic illustration of the role of Notch4 in the proliferation, apoptosis and migration of LUAD cells. Hypoxia induces the expression of Notch4. Increased level of Notch4 promotes cell proliferation, migration, and apoptosis resistance in A549 and H1299 cells. Hypoxia activates Notch4 via ERK/JNK/P38 MAPK signaling pathways to promote lung adenocarcinoma progression and metastasis. LUAD, lung adenocarcinoma; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Migration, Expressing, Protein-Protein interactions

Journal: European Journal of Histochemistry : EJH

Article Title: Epidermal growth factor-like domain multiple 7 (EGFL7): Expression and possible effect on biliary epithelium growth in cholangiocarcinoma

doi: 10.4081/ejh.2018.2971

Figure Lengend Snippet: A) Immunohistochemistry for EGFL7 in mucin-type iCCAs; cholangiocytes are almost completely negative for the expression of EGFL7 in all the representative picture (blue arrows); on the left, representative picture of a negative control; on the right, a higher magnification of an area to show the absence of immunoreactivity on biliary epithelium. B) Immunohistochemistry for EGFL7 in mixed-type iCCAs; cholangiocytes are significantly positive for the presence of EGFL7 in all the representative pictures (blue arrows); on the left, representative picture of a negative control; on the right, a higher magnification of an area to display the specific and enhanced immunoreactivity on biliary epithelium. Original magnification: 20x.

Article Snippet: Sections were then incubated overnight at 4°C with EGFL7 (1:100 Proteintech code 19291-1-AP).

Techniques: Immunohistochemistry, Expressing, Negative Control

Journal: Oncotarget

Article Title: Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β.

doi: 10.18632/oncotarget.6456

Figure Lengend Snippet: Figure 1: IL-1β modulates the expression of components of the Notch pathway in human aortic endothelial cells (HAECs). Confluent primary HAECs were treated with IL-1β (10 ng/ml) for 6 h or left untreated. A. mRNA levels (Sybr Green method, as reported in the Methods section) for Notch ligands were normalized with respect to β-actin and are expressed as the cDNA copy number (x103) (see Methods section). B. mRNA levels (Sybr Green method) for the Notch receptors Notch1, Notch2, Notch4 and for the Notch target genes Hes1 and Hey1 were normalized with respect to β-actin and are expressed as the cDNA copy number (x103) (see Methods section). A. and B.: Mean±SD. C. Representative western blot showing levels of Jagged1, full-length Notch1 (FL) and the Notch1 intracellular domain (Notch1ICD) (left), Notch2ICD and Notch4ICD (right) in HAECs treated for 6 h with IL-1β or left untreated. β-Actin is the loading control. D. mRNA levels for Jagged1 in HAECs treated with IL-1β or left untreated for the reported times, quantified by the 2(-∆∆Ct) method (see Methods section) after normalization with respect to β-actin, expressed as a fold-change with respect to untreated sample at 0.5 h (100 arbitrary units). Mean±SEM. E. Representative western blot showing levels of Jagged1 in HAECs treated with IL-1β for the reported times. β-actin is the loading control. All experiments were performed in duplicate and repeated independently at least 3 times. *P < 0.05, **P < 0.01, ***P < 0.001, t-test.

Article Snippet: Proteins (40 μg) were run on SDS-polyacrylamide gels and blotted onto nitrocellulose membranes, which were blocked by incubation with 10% non-fat milk at room temperature for 1 h. The membranes were probed with primary antibodies (Jagged1 1:1000, Notch1 (bTAN) 1:1000, Notch2 1:1000 (DSHB, Iowa City, IA), Notch4 1:1000 (R&D Systems Europe, Ltd., Abingdon, UK), VCAM1 1:1000 and β-actin 1:20000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and an antibody against Notch1ICD cleaved at Val1744, recognizing both human and murine Notch1ICD (Cell Signaling, Beverly, MA)), by incubation overnight at 4°C with the appropriate antibody in TBS, 0.05% Tween 20, 5% milk.

Techniques: Expressing, SYBR Green Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β.

doi: 10.18632/oncotarget.6456

Figure Lengend Snippet: Figure 5: Notch1ICD-mediated VCAM1 induction is partly counteracted by NF-kB inhibition in human umbilical vein endothelial cells (HUVECs). HUVECs were transduced with either a retroviral vector co-expressing a flag-tagged murine Notch1ICD (Notch1ICD*) and the enhanced green fluorescent protein (eGFP) or an eGFP vector (EV*) as a control. After 48 h from infection, cells were subjected to pretreatment with the NF-kB inhibitor BAY 11-7082 (20 µM) for 1 h or with vehicle (DMSO) and they were then treated with IL-1β for 1 h or left untreated. A. Levels of VCAM1 mRNA were quantified by the 2(-∆∆Ct) method (see Materials and Methods section) after normalization with respect to β-actin, and expressed as a fold-change relative to EV*-vehicle-treated cells (1 arbitrary unit). Mean±SD. B. Representative western blot showing levels of intracellular domain (Notch1ICD), Jagged1 and VCAM1 in HUVECs treated as in A. α-tubulin is the loading control. The experiments were performed independently and repeated at least twice. **P < 0.01, ***P < 0.001, ANOVA (Bonferroni correction). C. Proposed model of Notch1-dependent induction of endothelial VCAM1. In endothelial cells, IL-1β reduces the transcription of the Notch4 gene in an NF-kB-dependent manner, leading to downregulation of both the active Notch4ICD form and expression of the Notch4 target gene Hes1. Both these both phenomena are responsible, at least in part, for the upregulation of VCAM1 in the endothelium. Concomitantly, IL-1β induces VCAM1 expression in an NF-kB-dependent manner. In parallel, IL-1β decreases Notch1 transcript levels and upregulates expression of the Notch ligand Jagged1 via a mechanism involving NF-kB. Jagged1 binds to Notch1, leading to the cleavage of the receptor and the sustained formation of the activated form Notch1ICD, in turn favoring Jagged1 expression. At the same time, Notch1ICD amplifies the NF-kB-dependent VCAM1 expression induced by IL-1β. Possibly due to Notch1-inducing VCAM1 upregulation in the absence of IL-1β, the activation of Notch1 also decreases Notch4 and Hes1 expression.

Article Snippet: Proteins (40 μg) were run on SDS-polyacrylamide gels and blotted onto nitrocellulose membranes, which were blocked by incubation with 10% non-fat milk at room temperature for 1 h. The membranes were probed with primary antibodies (Jagged1 1:1000, Notch1 (bTAN) 1:1000, Notch2 1:1000 (DSHB, Iowa City, IA), Notch4 1:1000 (R&D Systems Europe, Ltd., Abingdon, UK), VCAM1 1:1000 and β-actin 1:20000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and an antibody against Notch1ICD cleaved at Val1744, recognizing both human and murine Notch1ICD (Cell Signaling, Beverly, MA)), by incubation overnight at 4°C with the appropriate antibody in TBS, 0.05% Tween 20, 5% milk.

Techniques: Inhibition, Transduction, Retroviral, Plasmid Preparation, Expressing, Control, Infection, Western Blot, Activation Assay

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: 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