Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not alter the steady-state levels of the NOTCH3 WT or C185R protein. A , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells with and without doxycycline-inducible expression of RFNG. NOTCH3 and RFNG were detected by antibodies against human NOTCH3 extracellular domain (ECD) and human RFNG. TUBULIN was blotted with an antibody against human/mouse α-tubulin as a loading control. The white and black arrowheads indicate full-length NOTCH3 and the ECD, respectively. B , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells treated with or without 0, 0.1, 0.5, or 1 μg/ml doxycycline. RFNG was detected by an antibody against HA, which tagged RFNG. The black arrowheads indicate N3ECDs. C , relative amounts of N3ECD WT and C185R with or without RFNG expression, as determined by Western blotting (B) (independent biological replicates N = 3). N3ECD band intensities were normalized to those observed without doxycycline. D , representative Western blot of cell lysates collected from cultured N3WT-RF-HeLa and N3C185R-RF-HeLa cells with or without RFNG expression and treated with puromycin for 0 or 16 h. The black arrowheads indicate N3ECDs. E , relative band intensities of N3ECD at 16 h normalized to those observed at 0 h, as determined by Western blotting in (D) (independent biological replicates N = 3). Data information: In C and E , data are presented as mean ± SD. n.s., not significant (C: Tukey’s post hoc test following one-way ANOVA; E: Tukey’s post hoc test following two-way ANOVA). HA, hemagglutinin; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Western Blot, Cell Culture, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG enhances JAG1-induced degradation of NOTCH3 WT but not that of R141C and C185R. A , images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/C185R-RF-HeLa with MIG-3T3 or JAG1-3T3 cells. N3WT/C185R-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The left panel shows GFP expressed in MIG-3T3 and JAG1-3T3 ( green ). The middle panel shows N3ECD ( magenta ). The right panel shows an overlay. Arrows indicate N3ECD transendocytosed into JAG1-3T3. The scale bar represents 10μm. B , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and C185R (independent biological replicates, N = 4). C , representative images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/R141C-RF-HeLa with JAG1-3T3 cells. N3WT/R141C-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The scale bar represents 10μm. D , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and R141C (independent biological replicates, N = 5). Data information: In B and D , data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, n.s., not significant (Tukey’s post hoc test following three-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not affect JAG1-mediated transendocytosis of NOTCH3 WT and C185R. A , schematic representation of the immunocytochemistry workflow used to separately detect NOTCH3 present in the cell membrane and in JAG1-3T3 cells. After N3WT/C185R-RF-HeLa cells transfected with NOTCH2 siRNA were cocultured with JAG1-3T3 cells, the fixed cells were stained with two different anti-NOTCH3ECD (N3ECD) antibodies (clones 1G5 and BAF1559) before and after cell permeabilization, respectively. The scale bars indicate 20 μm. B , representative confocal images of cocultured N3WT/C185R-RF-HeLa and JAG1-3T3 cells, as depicted in (A). The white arrowheads indicate NOTCH3 particles detected by antibodies against N3ECD (clones 1G5 and BAF1559), indicating the presence of NOTCH3 on the cell membrane. The scale bars indicate 20 μm. C , the ratio of NOTCH3 present on the cell membrane against the total NOTCH3 in JAG1-3T3 cells. Box plots: 10 to 90 percentile. (independent biological replicates: N = 3; total cell numbers, WT RFNG-: n = 86, WT RFNG+: n = 86, C185R RFNG-: n = 93, C185R RFNG+: n = 81). ∗∗p < 0.01, ∗∗∗∗ p < 0.0001 (Tukey’s post hoc tests following two-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Membrane, Transfection, Staining, Clone Assay

Journal: QJM: An International Journal of Medicine

Article Title: Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke

doi: 10.1093/qjmed/hcae136

Figure Lengend Snippet: Biomarker discovery by quantitative proteomics. ( A ) Plasma microvesicle isolation was carried out based on the vascular event outcomes of ischemic stroke patients, following a specific workflow. ( B ) A heatmap was generated to display the differentially upregulated proteins in the Event+ and Event− groups, respectively, where P < 0.05. ( C ) The differentially expressed proteins underwent pathway analysis using the PANTHER Classification System. ( D ) The Genotype-Tissue Expression (GTEx) portal revealed that Notch3 is predominantly expressed in arterial tissues.

Article Snippet: We first sought to validate our previous findings using a commercial ELISA assay (Human NOTCH3 ELISA kit, Cusabio) on plasma samples from the same group of patients.

Techniques: Biomarker Assay, Isolation, Generated, Expressing

Journal: QJM: An International Journal of Medicine

Article Title: Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke

doi: 10.1093/qjmed/hcae136

Figure Lengend Snippet: Plasma NOTCH3 predicts cardiovascular recurrence in men with ischemic stroke. NOTCH3 protein levels were measured directly in plasma samples of ( A ) the discovery cohort comparing levels among ischemic stroke patients with and without events (Event+ and Event−, respectively), and in ( B ) a larger validation cohort comparing plasma NOTCH3 in ischemic stroke patients with and without events (Event+ and Event−, respectively) and age-matched healthy controls. ( C ) The Kaplan–Meier plot shows survival probability of men with ischemic stroke according to quartiles of plasma NOTCH3 (log-rank P values = 0.020) suggesting that individuals in the highest quartile (>1600 pg/ml) of plasma NOTCH3 are more likely to experience cardiovascular recurrence. Statistical analysis was performed using Student’s t -test with Bonferroni adjustments, where * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: We first sought to validate our previous findings using a commercial ELISA assay (Human NOTCH3 ELISA kit, Cusabio) on plasma samples from the same group of patients.

Techniques:

Journal: QJM: An International Journal of Medicine

Article Title: Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke

doi: 10.1093/qjmed/hcae136

Figure Lengend Snippet: Comparison in demographic, stroke characteristics, risk factors, laboratory investigations and cardiovascular events in men and women ^a

Article Snippet: We first sought to validate our previous findings using a commercial ELISA assay (Human NOTCH3 ELISA kit, Cusabio) on plasma samples from the same group of patients.

Techniques: Comparison, Cell Counting, Biomarker Assay

Journal: QJM: An International Journal of Medicine

Article Title: Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke

doi: 10.1093/qjmed/hcae136

Figure Lengend Snippet: The hazard ratio (HR) and 95% confidence intervals (CI) of cardiovascular recurrence were analyzed based on plasma NOTCH3 quartiles a

Article Snippet: We first sought to validate our previous findings using a commercial ELISA assay (Human NOTCH3 ELISA kit, Cusabio) on plasma samples from the same group of patients.

Techniques:

Journal: QJM: An International Journal of Medicine

Article Title: Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke

doi: 10.1093/qjmed/hcae136

Figure Lengend Snippet: NOTCH3 expression shows increased levels in mouse models of stroke and atherosclerosis. ( A ) Serial densitometric analysis and quantification of NOTCH3 protein in mouse serum following middle cerebral artery occlusion revealed a peak in NOTCH3 levels at 24 h, which continued to persist up to 72 h after cerebral reperfusion. Two technical replicates and pooled serum from six mice. ( B ) Representative immunofluorescence images of the innominate arteries of 22-week-old wild-type mice and Apoe-/- mice with advanced atherosclerotic plaque were examined. The images show NOTCH3-expressing endothelial cells in association with the plaque region. White arrowheads were used to indicate the location of these NOTCH3-expressing endothelial cells ( n = 3; L, lumen; P, plaque region). The scale bar represents 20 µm. ( C ) A quantification was performed to assess the number of NOTCH3-expressing endothelial cells in 14–20 analyzed arterial sections across three wild-type mice compared to three Apoe-/- mice. Data represent mean ± standard deviation; one-way ANOVA analysis; ** P < 0.01; *** P < 0.001; ns, non-significant.

Article Snippet: We first sought to validate our previous findings using a commercial ELISA assay (Human NOTCH3 ELISA kit, Cusabio) on plasma samples from the same group of patients.

Techniques: Expressing, Immunofluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Mechanics-activated fibroblasts promote pulmonary group 2 innate lymphoid cell plasticity propelling silicosis progression

doi: 10.1038/s41467-024-54174-5

Figure Lengend Snippet: a Volcano plot of differentially expressed genes in bulk RNA-Sequencing dataset of soft (~1 kPa) or stiff (~25 kPa) gel cultured fibroblasts. Upregulated genes and downregulated genes are highlighted in red and blue ( n = 3 biological replicates). b GO analysis of upregulated genes in stiff gel cultured fibroblasts predicts the involved biological processes. c KEGG enrichment analysis of upregulated genes. d Bioinformatics analysis of the protein–protein interaction network using STRING. e Western blot showing Notch3 intracellular domain (ICD) expression in primary lung fibroblasts cultured on different stiffness matrix gels. Uncropped blots are in source data. The bar graph compares Notch3 intracellular domain (ICD) expression in soft (~1 kPa) or stiff (~25 kPa) gel cultured fibroblasts ( n = 3 biological replicates). f Immunofluorescence images showing Notch3 ICD staining in MLg2908 and primary lung fibroblasts cultured on soft (~1 kPa) or stiff (~25 kPa) matrix gels. DAPI was used to label nuclei. Scale bar = 20 μm. Bar graphs compare Notch3 ICD fluorescence intensities ( n = 7 biological replicates, MLg2908; n = 4 biological replicates, primary fibroblasts). g Schematic showing Notch3 flox mouse construction, and the breeding strategy for fibroblast-specific Notch3 depletion mice. Lung primary fibroblasts were then isolated for in vitro experiments. h Immunofluorescence images showing pro-IL-18 staining in primary lung fibroblasts isolated from Notch3 fl/fl and Notch3 fl/fl Col1a2 -creERT littermates cultured on stiff (~25 kPa) matrix gel. Scale bar = 20 μm. The bar graph compares pro-IL-18 fluorescence intensity of Notch3 fl/fl and Notch3 fl/fl Col1a2 -creERT primary lung fibroblasts cultured on stiff (~25 kPa) matrix gel ( n = 7 biological replicates). i Flow histogram showing primary lung fibroblast caspase 1 activity (FLICA). The bar graph compares FLICA MFI values in Notch3 fl/fl and Notch3 fl/fl Col1a2 -creERT primary lung fibroblasts cultured on stiff (~25 kPa) matrix gel. ( n = 5 biological replicates). j Bar graph comparing IL-18 levels in culture medium from stiff (~25 kPa) matrix gel stimulated primary lung fibroblasts isolated from Notch3 fl/fl and Notch3 fl/fl Col1a2 -creERT littermates as detected by ELISA ( n = 5 biological replicates). Bar graphs represent the combined results of three biologically independent experiments with similar results. Individual values are plotted in graphs. Data are shown as the mean ± SEM. P values were calculated using two-tailed unpaired Student’s t -tests and are indicated in graphs. Source data are provided in a source data file.

Article Snippet: Cells were permeabilized in 0.5% Triton X-100 in PBS for 15 min, then stained overnight at 4 °C with anti-αSMA (Cat: 14395-1-AP, 1:100, Proteintech), Notch3 (A-6, Cat: sc-515825, 1:50, Santa Cruz Biotechnology), and IL-18 (Cat: 10663-1-AP, 1:70, Proteintech) antibodies, all diluted in PBS plus 1% FCS.

Techniques: RNA Sequencing, Cell Culture, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Isolation, In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Mechanics-activated fibroblasts promote pulmonary group 2 innate lymphoid cell plasticity propelling silicosis progression

doi: 10.1038/s41467-024-54174-5

Figure Lengend Snippet: a Flow plot showing fibroblasts (CD45 – CD140a + CD90.2 + ) in CS-treated lungs. Flow histogram and bar graph compare Notch3 MFI values in fibroblasts from saline or CS-treated lungs (saline, n = 4 biological replicates; CS 8–10 weeks, n = 8 biological replicates). b Schematic showing tamoxifen (TAM)-induced fibroblast-specific Notch3 depletion. TAM was applied at early fibrogenesis stages (week 3–4 after CS treatment). c Flow plots showing NKp46 + (ILC1) and ST-2 + (ILC2) percentages in lungs ( n = 5 biological replicates). Bar graphs compare ILC2 and ILC1 percentages in ILCs and ILC1/ILC2 ratios in lungs. d Masson’s staining showing lung section images of whole lung lobes and zoomed in areas evaluating the degree of pulmonary fibrosis at weeks 8–10 and 12–14 or saline-treated counterparts. Scale bar = 600 μm (upper panel), 200 μm (middle panels), and 50 μm (lower panels). Bar graph compares fibrotic area percentages in sections ( n = 5 biological replicates). e Western blot showing FN and Col1 levels in lungs. Uncropped blots are in source data. The bar graph compares relative protein levels in mouse lungs ( n = 4 biological replicates). f Hydroxyproline (HYP) content in mouse lungs. The bar graph compares HYP content in lungs ( n = 4 biological replicates). g Schematic showing TAM-induced fibroblast-specific Notch3 depletion. TAM was applied at fibrosis progression stage (week 8–9 after CS treatment). h Flow plots showing NK1.1 + (ILC1) and ST-2 + (ILC2) percentages in CS-treated mouse lungs ( n = 5 biological replicates). Bar graphs compare ILC2 and ILC1 percentages in ILCs and ILC1/ILC2 ratios in lungs. i Schematic showing BLM-induced pulmonary fibrosis and TAM-induced fibroblast-specific Notch3 depletion. TAM was applied at the inflammatory stage (week 1–2 after BLM treatment). j Flow plots showing NKp46 + (ILC1) and ST-2 + (ILC2) percentages in BLM-treated mouse lungs ( n = 5 biological replicates). Bar graphs compare ILC2 and ILC1 percentages in pulmonary ILCs and ILC1/ILC2 ratios in lungs. Bar graphs represent the combined results of four biologically independent experiments with similar results. Individual values are plotted in graphs. Littermate mice were used in experiments. Data are shown as the mean ± SEM. P values were calculated using one-way ANOVA followed by Tukey’s tests ( c , d , e , f ) or two-tailed unpaired Student’s t -tests ( a , h , j ). P values are indicated in graphs. Source data are provided in a source data file.

Article Snippet: Cells were permeabilized in 0.5% Triton X-100 in PBS for 15 min, then stained overnight at 4 °C with anti-αSMA (Cat: 14395-1-AP, 1:100, Proteintech), Notch3 (A-6, Cat: sc-515825, 1:50, Santa Cruz Biotechnology), and IL-18 (Cat: 10663-1-AP, 1:70, Proteintech) antibodies, all diluted in PBS plus 1% FCS.

Techniques: Saline, Staining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Mechanics-activated fibroblasts promote pulmonary group 2 innate lymphoid cell plasticity propelling silicosis progression

doi: 10.1038/s41467-024-54174-5

Figure Lengend Snippet: Mechanics-activated fibroblasts secreting IL-18through Notch3 signaling promoted ILC2 conversion to ILC1 related to silicosis progression. Figure was partially created in BioRender. Fan, Y. (2023) BioRender.com/h98u449.

Article Snippet: Cells were permeabilized in 0.5% Triton X-100 in PBS for 15 min, then stained overnight at 4 °C with anti-αSMA (Cat: 14395-1-AP, 1:100, Proteintech), Notch3 (A-6, Cat: sc-515825, 1:50, Santa Cruz Biotechnology), and IL-18 (Cat: 10663-1-AP, 1:70, Proteintech) antibodies, all diluted in PBS plus 1% FCS.

Techniques:

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Sequence of primers used in the PCR assays

Article Snippet: The cells were then incubated with ammonium chloride (100 mM: Sigma) at room temperature for 10 min and they were then stained overnight with primary antibodies against Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany), Notch2 (BioLegend, Barcelona, Spain), Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) and Giantin (Biolegend, Barcelona, Spain), all diluted 1:100.

Techniques: Sequencing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: 2OHOA inhibits Notch signaling pathway. a Representative immunoblots of Notch2/3 FL (full length), Notch2/3 TM (transmembrane domain) and NICD2/3 (Notch intracellular domain) proteins after exposure of U-87 MG cells to 2OHOA (200 µM) for 6, 12, 24 and 48 h (four independent experiments with two replicates each). b The Hes1 protein levels after exposure to 2OHOA (200 µM) for 48 h as an indicator of its pharmacological effect in U-87 MG cells. c The mRNA expression of JAGGED1 , NOTCH1 , NOTCH2 , NOTCH3 , HES1 and CD3 genes in U-87 MG treated for 48 h with 2OHOA (200 µM) or the vehicle alone. The values are presented as the mean ± SEM of at least four independent experiments analyzed in triplicate. d JAGGED1 mRNA levels in U-87 MG cells exposed to 2OHOA (200 µM) or the vehicle alone for 72 h. The results are from three independent experiments with three replicates each and they are expressed as the % of the control ± SEM. e Cell localization of Notch3 (left panels) and Hes1 (right panels) in U-87 MG control cells (row 1, 2) or those exposed to 2OHOA (400 µM) for 48 h (row 3, 4). Images were taken at 40× magnification. Scale bar: 20 μm (row 1, 3), 5 μm (row 2,4). f Nuclear fluorescence intensity of Notch3 (upper graph) or Hes1 (lower graph) in two independent experiments. The statistical analysis was performed with a t -test with Welch’s correction relative to the untreated cells: *** p < 0.001

Article Snippet: The cells were then incubated with ammonium chloride (100 mM: Sigma) at room temperature for 10 min and they were then stained overnight with primary antibodies against Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany), Notch2 (BioLegend, Barcelona, Spain), Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) and Giantin (Biolegend, Barcelona, Spain), all diluted 1:100.

Techniques: Western Blot, Expressing, Control, Fluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Graphical illustration describing the 2OHOA mechanism of action to inhibit Notch2 and Notch3 signaling. Model for activated Notch2/3 signaling (left panel). Proposal model for 2OHOA mechanism of action (right panel) where its interaction with the furin enzyme prevents Notch2 processing in Golgi. Instead, Notch3 is transcriptionally repressed probably by indirect 2OHOA mechanism (indicated as dashed line). In addition, HES1 overexpression partially inhibits the 2OHOA effect on viability (narrower dashed line). Continuous lines indicate activation while transparent continuous lines represent lower activation of the pathway

Article Snippet: The cells were then incubated with ammonium chloride (100 mM: Sigma) at room temperature for 10 min and they were then stained overnight with primary antibodies against Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany), Notch2 (BioLegend, Barcelona, Spain), Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) and Giantin (Biolegend, Barcelona, Spain), all diluted 1:100.

Techniques: Over Expression, Activation Assay