Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Time- and dose-dependent inhibition of NAC on the intracellular domain of Notch3 (N3IC), but not Notch1 (N1IC). HeLa cells were treated with NAC (2-10 mM) for 0-24 h. B. Dose-dependent inhibition by NAC (0-10 mM, 6 h) on the protein expression of N3IC in HeLa cells. C. NAC treatment (5 mM, 0-12 h) reduces protein levels of N3IC and extracellular domain of Notch 3 (N3EC) but not full length Notch 3 precursor (N3FL) in HeLa cells. Densitometry quantifications of the protein bands were shown after normalization with their respective β-actin levels. Data are presented as means ± SE, n=3. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (2-10 mM, 0-24 h) decreases Hes1 and HRT1 protein levels in HeLa cells. B. NAC treatment (0-10 mM for 6 h or 5 mM for 0-12 h) decreases Hes1 and HRT1 mRNA expression in HeLa cells. The mRNA expression of NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. C. NAC treatment (0-10 mM, 12 h) inhibits Hes1 reporter activity in HeLa cells. The luciferase activity in NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. D. Notch3 siRNA knockdown reduces Hes1 and HRT1 levels in HeLa cells. Protein levels were determined 2 days after siRNA transfection. siCtrl, scramble siRNA; siNotch3, Notch3 siRNA. Protein densitometry quantifications were shown after normalization with β-actin levels. Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Activity Assay, Luciferase, Knockdown, Transfection

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Pre-treatment with a γ-secretase inhibitor, DAPT (20 μM, 30 min), had no effect on NAC-induced (5 mM, 0-12 h) decrease in N3IC protein expression in HeLa cells. B. NAC treatment (5 mM, 0-12 h) did not affect Notch3 mRNA expression in HeLa cells. C. Pre-treatment with NH 4 Cl (25 mM, 1 h), but not lactacystin (10 μM, 30 min), reversed NAC-induced (5 mM, 0-12 h) decrease of N3IC protein levels in HeLa cells. D. NAC treatment did not affect levels of exogenously expressed Notch3 active intracellular domain (N3ICD). HeLa cells were transfected with vectors expressing N3ICD or N3FL for 24 h, followed by treatment with NAC (5 mM, 0-12 h). E. Subcellular analysis of Notch3 protein levels following NAC treatment (5 mM, 6 h) in HeLa cells. Protein levels of N3FL, N3EC and N3IC in cytosolic, nuclear and membrane fractions were determined. Successful fractionation was evidenced by using the marker proteins GAPDH, cyclin B1, and Na + , K + -ATPase. N3FL, N3EC and N3IC denoted Notch3 full length, extracellular domain and intracellular domain, respectively. Protein densitometry quantifications were shown after normalization with β-actin (A, C, D) or their respective cellular compartment markers (E). Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Transfection, Membrane, Fractionation, Marker

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: N3ICD overexpression rescues NAC-induced inhibition of proliferation (A), migration (B), and invasion (C). A. Numbers of EV- and N3ICD-transfected cells were counted at 12-48 h after NAC treatment (0-10 mM, left panel). *, p < 0.05 compared with the EV-transfected cells within the same treatment and time point. B. Results of the wound healing assay (left panels) were expressed as the migration index (the distance migrated relative to the initial scraped gap) and that of EV-transfected cells without NAC treatment was set as 100% (middle panel). C. Cells per field on the insert membrane were imaged (left panels) and counted (middle panel). B and C: *, p < 0.05 compared with no NAC treatment; #, p < 0.05 compared with the EV-transfected cells within the same treatment. Percent rescue (A-C, right panels) after N3ICD expression was calculated by dividing the net change after NAC treatment in N3ICD-transfected cells by that in EV-transfected cells. Notch3 siRNA knockdown inhibits cell proliferation D. , migration E. , and invasion F. as assessed by the same approaches described above. Representative images for migration and invasion were shown. *, p < 0.05 compared with the siCtrl-transfected cells. All data are presented as mean ±SE, n=3. I, the initial seeded cell number. EV, empty vector; N3ICD, Notch3 active intracellular domain; siCtrl, scrambled siRNA; siNotch3, Notch3 siRNA.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Over Expression, Inhibition, Migration, Transfection, Wound Healing Assay, Membrane, Expressing, Knockdown, Plasmid Preparation

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (5 and 10 mM, 0-24 h) decreases N3IC protein levels in HCC1937 cells. Expression of exogenous N3ICD rescues NAC-induced inhibition of proliferation B. , migration C. , and invasion D. , and Notch3 siRNA knockdown inhibits proliferation E. , migration F. , and invasion G. in HCC1937 cells. Quantifications, sample size, statistics, and abbreviations for protein levels, proliferation, migration, and invasion assays were as described in Figure & legends.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Inhibition, Migration, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL

doi: 10.1084/jem.20161715

Figure Lengend Snippet: A13 Notch3 agonist antibody activates a ligand-insensitive Notch3 receptor with the C455R mutation in vitro. (A) Schematic diagram showing the structure of the Notch3 receptor and the binding site where the A13 antibody interacts with the NRR domain. Lin12-Notch (LNR), heterodimerization domain (HD), and transmembrane domain (TM) are shown. Luciferase reporter assay for Notch signaling in monocultures of HEK 293 cells expressing Notch3 WT receptor (B) or the Notch3 C455R (C) after treatment with A13 or control IgG antibodies. TET indicates concentration of tetracycline used to induce transgene expression. Luciferase reporter assay for Notch signaling in HEK 293 cell co-cultures. HEK 293 cells expressing Notch3 WT or C455R Notch3 receptors in co-culture with control cells (D) or with HEK 293 cells expressing Jagged 1 (E). HEK 293 cells expressing Notch3 WT in co-culture with control cells (F) or with cells expressing Jagged 1 (G) treated with IgG or A13 antibodies. HEK 293 cells expressing Notch3 C455R in co-culture with control cells (H) or with cells expressing Jagged 1 (I) treated with IgG or A13. ELISA measurements of N3ECD in cell culture supernatant for each culture, Notch3 WT (J) and C455R (K). Luciferase reporter assay for Notch signaling in monocultures of HEK 293 cells expressing Notch3 WT receptor in the presence of compound E (L). ELISA measurements of N3ECD in cell culture supernatant in the presence of compound E (M). Luciferase reporter assay for Notch signaling in monocultures of HEK cells expressing Notch3 WT receptor treated with increasing concentrations of A13 (N). ELISA measurements of N3ECD in cell culture supernatant from cells treated with increasing concentrations of A13 (O). Notch3 signaling activation measured via gene reporter assay correlated with increased levels of N3ECD in cell culture supernatants measured by ELISA (P). (B–P) *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were analyzed via ANOVA. Values in graphs are expressed as the means ± SEM. The results are representative of three independent experiments each including three technical replicates per condition.

Article Snippet: The recombinant human N3ECD, originally used to raise the antibodies (1559-NT-050; R&D Systems), was used as the standard, and a positive control.

Techniques: Mutagenesis, In Vitro, Binding Assay, Luciferase, Reporter Assay, Expressing, Control, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL

doi: 10.1084/jem.20161715

Figure Lengend Snippet: A13 Notch3 agonist antibody reverses plasma biomarker changes in N3ECD and endostatin/collagen 18α1 in CADASIL mice. Dot plots showing plasma levels of N3ECD (A), endostatin/collagen 18α1 (B), IGFBP-1 (C), and HTRA1 (D) in 6-wk-old C455R mice treated with the A13 ( n = 10) or IgG ( n = 8) antibodies from 10 independent rounds of injections. (A-D) **, P < 0.01. Statistical analysis was done via unpaired two-tailed Student's t test. Each data point represents the value for a single mouse. Horizontal lines show the means ± SEM. The C455R mice were littermates.

Article Snippet: The recombinant human N3ECD, originally used to raise the antibodies (1559-NT-050; R&D Systems), was used as the standard, and a positive control.

Techniques: Clinical Proteomics, Biomarker Discovery, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells

doi: 10.1074/jbc.M112.446633

Figure Lengend Snippet: Cytokine-dependent expression of NOTCH receptors, ligands, and target genes in rat NP cells. Real-time RT-PCR analysis showed selective induction in expression of NOTCH1 (A), NOTCH2 (B), and JAGGED2 (C) by NP cells treated with IL-1β and TNF-α. ctr: control, Hprt: hypoxanthine phosphoribosyltransferase. D, expression of NOTCH target genes HEY1, HEY2, and HES1 was induced by IL-1β and TNF-α treatment of NP cells and represented as fold change relative to corresponding untreated control (considered as 1). E, Western blot analysis shows that when treated with IL-1β or TNF-α, NP cells increase levels of cleaved NOTCH1, cleaved NOTCH2, and HEY2 but not of cleaved NOTCH3. Densitometric analysis of multiple blots from experiment (E) above showed that levels of cleaved NOTCH1 (F), cleaved NOTCH2 (G), and HEY2 (H). Values shown are mean ± S.E. of three independent experiments; *, p < 0.05.

Article Snippet: NOTCH1, NOTCH2, NOTCH3, p65, and IKKb antibodies were from Cell Signaling; Hey2 was from Peprotech; β-tubulin was from Developmental Studies Hybridoma Bank; and GAPDH was from Novus Biologicals.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Schematic cartoon showing the generation of pericyte (Per)- and smooth muscle cell (SMC)-specific CADASIL mice by crossing conditional Notch3 R170C (lox-STOP-lox) mice with ATP13A5-CreER (pericytes) or Myh11-CreER (SMCs). Tamoxifen (Tam) induces cell-specific expression of mutant Notch3. ( b ) RNAscope in situ hybridization of Notch3 mRNA (magenta) with lectin-stained vessels (white) in corpus callosum. Insets highlight cell-type-specific transcript accumulation (capillaries in Per-Notch3 R170C , arterioles in SMC-Notch3 R170C ). DAPI (blue), nuclei. ( c ) Quantification of Notch3 mRNA confirms mural-cell-specific induction. ( d ) Immunostaining of NOTCH3 extracellular domain (ECD, magenta) and endothelial CD31 (green). Arrows indicate NOTCH3 aggregates restricted to mural cells. ( e ) Quantitative analysis of NOTCH3 protein fluorescence shows selective mural-cell accumulation. In c) Each dot represents an individual vessel from N = 4–8 mice per group. In e) each dot represents a mouse. Box plots show median ± interquartile range; whiskers, min/max; Scale bars, 10 µm. One-way ANOVA with Tukey’s test, ***, P < 0.001, **, P < 0.01, ns = not significant).

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Expressing, Mutagenesis, RNAscope, In Situ Hybridization, Staining, Immunostaining, Fluorescence

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Schematic representation of parameters analyzed: vascular area fraction, branching density, total vessel length, and nearest neighbor distance. ( b ) Representative images of CD31-labeled cerebral vessels in the cortex from Per-Notch3 and SMC-Notch3 mice expressing WT or mutant Notch3 R170C . Scale bar = 50 µm. ( c–f ) Quantification of vascular parameters across brain regions (corpus callosum, cortex, hippocampus, dorsal striatum). Parameters include ( c ) vascular area fraction, ( d ) branching density, ( e ) vessel length density, and ( f ) nearest neighbor distance. Box plots show median ± interquartile range; each dot represents individual mice; whiskers indicate min/max. Statistical significance: *p < 0.05, **p < 0.01, ns = not significant.

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Labeling, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Representative images of extravascular fibrinogen (blue) and vascular marker CD31 (magenta) in cortical regions. Scale bar = 50 µm. ( b ) Quantification of extravascular fibrinogen immunostaining across corpus callosum, cortex, hippocampus, and dorsal striatum. ( c ) Representative images showing smooth muscle actin (SMA, green) coverage on CD31-labeled vessels (magenta) in corpus callosum regions. Scale bar = 50 µm. ( d ) Quantification of SMA coverage of blood vessels in Per-Notch3 R170C and SMC-Notch3 R170C mice compared to controls (*p < 0.05). Box plots show median ± interquartile range; each dot represents individual mice; whiskers indicate min/max. Statistical significance: *p < 0.05, ns = not significant.

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Marker, Immunostaining, Labeling

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Schematic illustrating analyzed brain regions: cortex, corpus callosum, hippocampus, and dorsal striatum. ( b ) Representative immunostaining for microglial markers Iba1 (green) and CD68 (magenta) showing increased microglial activation in corpus callosum of Per-Notch3 R170C mice and SMC-Notch3 R170C compared to littermate controls (Per-Notch3 WT and SMC-Notch3 WT ). Insets show magnified examples of microglial cells. Scale bars, 50 µm (overview), 20 µm (insets). ( c ) Quantification of Iba1 (upper row) and CD68 (lower row) fluorescence intensity (one-way ANOVA with Tukey’s test; P < 0.05, *P < 0.01, ns = not significant). Box plots show median ± interquartile range; each dot represents individual mice; whiskers indicate min/max.

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Immunostaining, Activation Assay, Fluorescence

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Representative images of MBP immunostaining in the corpus callosum (cc) of pericyte- and smooth muscle cell-specific Notch3 R170C mice and controls. Scale bar = 50 µm. ( b ) Quantification of MBP fluorescence intensity across corpus callosum, cortex, hippocampus, and dorsal striatum. Box plots show median ± interquartile range; each dot represents individual mice; whiskers indicate min/max. Statistical significance: ** p < 0.01, *p < 0.05, ns = not significant.

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Immunostaining, Fluorescence

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Schematic of workflow: brain tissue was collected, vessels were isolated, and subjected to quantitative LC-MS/MS proteomics. ( b ) Representative images of CD31-labeled brain vasculature confirming successful isolation of vascular fragments. ( c ) Western blot of isolated vessels shows accumulation of full-length NOTCH3 (∼90 kDa) in Per-Notch3 R170C and SMC-Notch3 R170C mice; GAPDH used as loading control. ( d ) Heatmap of differentially expressed proteins reveals distinct profiles between genotypes (Differentially expressed proteins were identified separately in each comparison; the proteins represented in each heatmap are not identical across the two models.) ( e ) Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) show clear separation of mutant and control groups. ( f ) Volcano plots of differentially expressed proteins (Per-Notch3 R170C vs WT and SMC-Notch3 R170C vs WT) highlighting top up- and down-regulated proteins, including shared hits LMO3 (up) and DGAT2 (down). ( g ) Venn diagram summarizing the overlap of most differentially expressed proteins (FDR < 0.05; FC > 0.5) between Per- and SMC-Notch3 R170C mice. ( h ) Gene set enrichment analysis (GSEA) of proteomic data reveals distinct pathway enrichment patterns: Per-Notch3 R170C mice show upregulation of mitochondrial respiration and protein folding pathways, while SMC-Notch3 R170C mice display enrichment in RNA processing, chromatin remodeling, and immune pathways. Data represent normalized enrichment scores (NES) for selected top-ranking gene sets.

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Labeling, Western Blot, Control, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Distribution of significantly up- and downregulated phosphopeptides in isolated brain vessels from pericyte-specific (Per-Notch3 R170C , left) and smooth muscle cell–specific (SMC-Notch3 R170C , right) mutant mice compared to respective Notch3 WT controls. Percentages indicate the proportion of phosphopeptides exhibiting increased or decreased phosphorylation. ( b ) Network analysis of differentially phosphorylated proteins in Per-Notch3 R170C mice highlights functional modules related to heat shock protein regulation (blue), mitochondrial redox function (red), and focal adhesion/cell junction function (green). ( c ) Network analysis of SMC-Notch3 R170C phosphoproteome reveals clusters associated with mRNA processing (green), spliceosome assembly (purple), and DNA repair (yellow). Node size reflects the number of connections; edges represent experimentally supported protein– protein interactions.

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Isolation, Mutagenesis, Phospho-proteomics, Functional Assay, Protein-Protein interactions

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Boxplot of relative abundance of canonical NOTCH3 pathway components measured by quantitative proteomics in pericyte- and SMC-specific Notch3 R170C mice and respective controls. ( b ) Network diagram of the NOTCH3 interactome displaying phosphoproteins quantified in pericytes (pink), SMCs (blue), both (green), or not identified (gray). ( c ) Dot plots showing relative abundance of phosphorylation at five sites along the NOTCH3 protein in pericytes (left) and SMCs (right), with schematic indicating phosphosite positions. ( d ) Network of proteins within the NOTCH3 interactome with successfully quantified protein-protein interactions (PPIs) shown in blue and non-identified proteins in gray. ( e ) Dot plots showing relative abundance of phosphorylation at three sites on Rbpj (Y65, T37, S176) across mural cell types and genotypes, with schematic indicating phosphosite locations.

Article Snippet: A conditional Notch3 R170C (Kozak mutation) knock-in mouse line was generated on a C57BL/6 background (Cyagen Biosciences) using CRISPR/Cas9-mediated genome engineering.

Techniques: Quantitative Proteomics, Phospho-proteomics, Protein-Protein interactions