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: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 1 c-Cbl ubiquitin ligase regulates proteasomal degradation of Notch3-IC. (a) Co-transfection of HEK293 cells with flag Notch3-IC and HA-c-Cbl expression plasmids. Whole lysates were immunoprecipitated with anti-flag antibody and revealed in western blot with anti-Ub and anti-HA antibodies. Anti-flag antibody was used as a control of the immunoprecipitation assay. Total lysates were analyzed for the expression of c-Cbl plasmid using anti-HA antibody. (b, c) Co-transfection of HEK293 cells with flag Notch3-IC and HA c-Cbl expression plasmids: cells were treated with (b) proteasome inhibitor MG132 in a time course study for 0–5 h or with (c) cycloheximide (CHX) and/or MG132 for 5 h before lysis. In both experiments, whole lysates obtained were revealed in western blot with anti-flag antibody, followed by anti-b-actin antibody used as a control of sample loading (left panels). The right panels show the relative quantification as determined by optical densitometry (OD).

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Ubiquitin Proteomics, Cotransfection, Expressing, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Lysis

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 3 Protein kinase C y (PKCy) regulates c-Cbl phosphorylation status in ex vivo and in vitro experiments, resulting in the regulation of Notch3-IC protein levels. (a) Whole-cell extracts of thymocytes from wt, tg N3-IC and N3-IC/pTa/ mice were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P- Ser PKC substrate antibody. Anti-c-Cbl immunoblotting was used as a control of the immunoprecipitation assay. Western blot analysis of c-Cbl in total lysates shows that the modification of c-Cbl phosphorylation is not accounted for by changes of its protein levels. Data are representative of three similar experiments. (b) Treatment of N3-IC/pTa/ double-mutant thymocytes with phorbol 12-myristate 13-acetate (PMA) for 0–15–30 min before lysis: total lysates were revealed in western blot with anti-P-tyr and anti-Cbl antibodies. Molecular weight markers are shown on the left (left panel). The same samples were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P-Ser PKC substrate and anti-PKCy antibodies. Anti-c-Cbl immunoblotting was used as a control of the immunoprecipitation assay (right panel). (c) Treatment of N3-IC/ pTa/ double-mutant thymocytes with PMA for 3 h before lysis: western blot analysis of Notch3 protein levels. Anti-b-actin immunoblotting was used as a control of sample loading. (d) Co-transfection of HEK293 cells with HA-c-Cbl plasmid in presence or absence of DN-PKCy and CA-PKCy expression vectors. Whole lysates were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P-Ser and anti-PKCy antibodies. The immunoblotting with anti-Cbl antibody was used as a control of the immunoprecipitation assay. (e) Co-transfection of HEK293 cells with flag Notch3-IC and HA c-Cbl plasmids in presence or absence of DN-PKCy and CA-PKCy expression vectors. Whole lysates were immunoprecipitated with anti-flag antibody and revealed in western blot with anti-Ub antibody, followed by anti-flag antibody used as a control of the immunoprecipitation assay. The same whole lysates were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-flag antibody to detect c-Cbl/Notch3 interaction. (f) HEK293 cells, co-transfected as previously described, were treated with proteasome inhibitor MG132 for 5 h before lysis. Whole lysates were revealed in western blot with anti-flag antibody, followed by anti-b-actin antibody, used as a control of sample loading.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Phospho-proteomics, Ex Vivo, In Vitro, Immunoprecipitation, Western Blot, Control, Mutagenesis, Lysis, Molecular Weight, Cotransfection, Plasmid Preparation, Expressing, Transfection

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 4 pTa directly interacts with Notch3 and regulates its recruitment to lipid rafts. (a) Whole-cell extracts, cytosol and membrane fractions and rafts and nonrafts fractions of N3-232T cells were revealed in western blot with anti-N3EC and anti-N3IC antibodies. Results are representative of three similar experiments. (b) Left panel: confocal microscopy of thymocytes from wt and tg N3-IC mice stained with Alexa Fluor 594-conjugate CTB and rabbit polyclonal anti-Notch3 antibody, as described in Materials and methods section. Right panel: rafts and nonrafts fractions derived from the same cells were revealed in western blot with anti-N3EC and anti-pTa antibodies. Anti-ZAP-70 was used to control the activation status. For T-cell activation, cells were incubated with anti-CD3 antibody as described in Materials and methods section. (c) Rafts and nonrafts fractions derived from SCB29 and SCIET27 pre-T-cell lines were revealed in western blot with anti-N3EC antibody. (d) Whole-cell extracts (left panel), rafts and nonrafts fractions (right panel) derived from tg N3-IC and N3-IC/pTa/ mice were revealed in western blot with anti-N3EC. (e) Co-transfection of HEK293 cells with HA- Notch3-IC and pTa expression plasmids. Whole lysates were immunoprecipitated with anti-pTa antibody and revealed in western blot with anti-HA antibody, followed by anti-pTa antibody, used as a control of the immunoprecipitation assay. * indicates a nonspecific band (left panel). Whole lysates from 2017 pre-T-cell line were immunoprecipitated with anti-pTa antibody and revealed in western blot with anti-N3IC antibody, followed by anti-pTa antibody, used as a control of the immunoprecipitation assay (right panel). Data are representative of three similar experiments. Anti-p56Lck and anti-tubulin immunoblotting were used as a control of fractionation; anti-b-actin immunoblotting was used to monitor sample loading. WCE, whole-cell extracts; C, cytosol; M, membrane; R, rafts; NR, nonrafts; FL, full-length receptor; EC, extracellular fragment; TM, transmembrane fragment; IC, intracellular fragment; un, untransfected cells; tr, transfected cells.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Membrane, Western Blot, Confocal Microscopy, Staining, Derivative Assay, Control, Activation Assay, Incubation, Cotransfection, Expressing, Immunoprecipitation, Fractionation, Transfection

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 5 Notch3/pTa relationship favors c-Cbl recruitment to lipid rafts. (a) Rafts and nonrafts fractions from wt thymocytes, resting or activated with anti-CD3 antibody, and tg N3-IC thymocytes, not activated, were revealed in western blot with anti c-Cbl antibody. Anti-ZAP-70 and anti-b-actin immunoblotting were used to control the activation process and to monitor sample loading, respectively. (b) Treatment of N3-232T cells with furine-like inhibitor A23187 before rafts isolation. Rafts and nonrafts fractions were revealed in western blot with anti-c-Cbl antibody. Anti-N3EC and anti-N3IC immunoblotting were used as a control of the treatment; anti-p56Lck and anti-Lat immunoblotting were used as a control of rafts aggregation process. * indicates the Notch3-IC protein. (c) Rafts and nonrafts fractions from tg N3-IC and N3-IC/pTa/ thymocytes were revealed in western blot with anti-c-Cbl antibody. Anti-p56Lck and anti-tubulin immunoblotting were used as a control of fractionation. The samples are normalized using b-actin protein expression (left panel). The right panel shows the quantification of c-Cbl protein associated to rafts in tg N3-IC and N3-IC/pTa/ thymocytes. Data represent the average of three independent experiments. (d) c-Cbl/Notch3-IC cytosolic interaction after rafts disrupting. Treatment of N3-232T cells with methyl-b-cyclodextrin (MbCD) before subcellular fractionation: cytosolic fractions derived from N3- 232T MbCD-treated and -untreated cells were used for immunoprecipitation assay with anti-c-Cbl antibody and revealed in western blot with anti-N3IC antibody, followed by anti-c-Cbl antibody, used as a control of the immunoprecipitation assay. R, rafts; I, intermediate fractions; NR, nonrafts; FL, full-length receptor; EC, extracellular fragment; TM, transmembrane fragment.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Western Blot, Control, Activation Assay, Isolation, Fractionation, Expressing, Derivative Assay, Immunoprecipitation

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 6 Hypothetical model linking c-Cbl different subcellular localization with Notch3 degradation. (a) In presence of a functional pre-TCR/Notch3 relationship, c-Cbl is able to translocate to the rafts compartment, where it is preferentially phosphorylated in serine- rich motifs through protein kinase C y (PKCy). In these conditions c-Cbl seems to lack its E3 ubiquitin ligase upon Notch3, thus sustaining the oncogenic role of Notch3 and pTa relationship with respect to T-cell leukemogenesis. (b) In the absence of pre-TCR, c-Cbl localizes preferentially in cytosolic fraction. In addition, deletion of pTa results in reduction of PKCy activity that in turn favors tyrosine phosphorylation of c-Cbl and its increased E3 ubiquitin ligase activity upon Notch3-IC, targeting it to proteasomal-degradative pathway and contributing to the rescue of T-cell leukemia. P-tyr, tyrosine phosphorylation; P-Ser, serine phosphorylation; Ub, ubiquitination.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Functional Assay, Ubiquitin Proteomics, Activity Assay, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: NOTCH3 alterations across human cancers and CRC. (a) NOTCH3 mRNA expression levels across TCGA cancer types green represents normal and red is cancer. The red labels indicate up-regulation in corresponding tumor type and the green labels mean down-regulation (statistically significant). (b) Distribution of NOTCH3 somatic mutations (lollipop plot showing mutation frequency and type across protein domains); left, pan-cancer; right, CRC (N = 534). (c) Negative correlation between NOTCH3 expression and cg06650786 methylation (scatter plot with Spearman R=-0.42, p=1.45e-302, N = 10967). (d) NOTCH3 expression stratified by copy number variation (N = 526). The x-axis is copy number of NOTCH3 across CRC samples and y-axis is NOTCH3 expression. (e) Combined alteration frequency (mutations + CNVs) across cancer (bar plot with CRC ranked 7th at 7%) patients (N = 10967).

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Expressing, Mutagenesis, Methylation

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: NOTCH3 expression predicts poor survival across cancers. (a) Forest plots of Cox regression for NOTCH3 association with (a) overall survival (OS) and (b) progression-free survival (PFS) in multiple cancers (HR >1 indicates worse prognosis) using Cox univariate regression. (c, d) Comparing high vs. low NOTCH3 expression groups for (c) OS and (d) PFS, showing pvalue and HR. Sample size: Acute Myeloid Leukemia: 161; Adrenocortical Carcinoma: 78; Bladder Urothelial Carcinoma: 406; Brain Lower Grade Glioma: 513; Breast Invasive Carcinoma: 1082; Cervical Squamous Cell Carcinoma: 294; Cholangiocarcinoma: 36; Colorectal Adenocarcinoma: 588; Diffuse Large B-Cell Lymphoma: 48; Esophageal Adenocarcinoma: 181; Glioblastoma Multiforme: 154; Head and Neck Squamous Cell Carcinoma: 514; Kidney Renal Clear Cell Carcinoma: 510; Kidney Renal Papillary Cell Carcinoma: 282; Liver Hepatocellular Carcinoma: 365; Lung Adenocarcinoma: 501; Lung Squamous Cell Carcinoma: 478; Mesothelioma: 86; Ovarian Serous Cystadenocarcinoma: 299; Pancreatic Adenocarcinoma: 177; Prostate Adenocarcinoma: 493; Sarcoma: 253; Skin Cutaneous Melanoma: 426; Stomach Adenocarcinoma: 407; Thymoma: 118; Thyroid Carcinoma: 497; Uterine Carcinosarcoma: 57; Uterine Corpus Endometrial Carcinoma: 526; Uveal Melanoma: 80. (e) Overall (top) and progression-free (bottom) survival curve of NOTCH3-low and NOTCH3-high samples in TCGA-CRC dataset. The bottom represents the cancer types (N = 588). (f) Area under the receiving operating characteristic (AUROC) curves for 1/3/5-year survival prediction using NOTCH3 expression across cancers. (Same sample size as A-D) (g) MSI score differences between NOTCH3 mutant vs. wild-type CRC.

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Expressing, Mutagenesis

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: NOTCH3 alteration enhance immune infiltration in CRC. (a) Heatmap of immune cell abundance differences in NOTCH3-mutant vs. wild-type tumors across cancers. Blue corresponds to diminished infiltration in NOTCH3-mutated tumors and red represent increased. Only the representative cell types indicated in the right part of the figure. Sample size sued listed as below: Acute Myeloid Leukemia: 174, Adrenocortical Carcinoma: 79, Bladder Urothelial Carcinoma: 408, Brain Lower Grade Glioma: 515, Breast Invasive Carcinoma: 1083, Cervical Squamous Cell Carcinoma: 295, Cholangiocarcinoma: 37, Colorectal Adenocarcinoma: 593, Diffuse Large B-Cell Lymphoma: 49, Esophageal Adenocarcinoma: 182, Glioblastoma Multiforme: 161, Head and Neck Squamous Cell Carcinoma: 516, Kidney Renal Clear Cell Carcinoma: 511, Kidney Renal Papillary Cell Carcinoma: 284, Liver Hepatocellular Carcinoma: 367, Lung Adenocarcinoma: 511, Lung Squamous Cell Carcinoma: 485, Mesothelioma: 88, Ovarian Serous Cystadenocarcinoma: 301, Pancreatic Adenocarcinoma: 178, Prostate Adenocarcinoma: 494, Sarcoma: 254, Skin Cutaneous Melanoma: 444, Stomach Adenocarcinoma: 413, Thymoma: 120, Thyroid Carcinoma: 499, Uterine Carcinosarcoma: 58, Uterine Corpus Endometrial Carcinoma: 528, Uveal Melanoma: 81 (b) Immune infiltration difference in CRC (boxplot showing infiltration abundances in wild-type and mutant samples). (c) Normalized cell proportion difference between NOTCH3-high vs. -low groups. (d) Expression of NOTCH3 across cell types, where dot size represents percentage of cells expression NOTCH3 and the color indicates average expression value. (e) Immune cell proportion difference between NOTCH3-low and NOTCH3-high samples. (f) Correlation between NOTCH3 expression and immune cell proportions. The x-axis is the cell proportion and y-axis is log2 transformed NOTCH3 expression (g) Expression of NK and CD8+T cell marker in NOTCH3-low and NOTCH3-high samples in scRNA-seq data. (h) PVR expression and NOTCH3 expression in scRNA-seq samples, each dot represents a sample (N = 62), corresponding to the expression of PVR (x axis) and NOTCH3 (y-axis). **p < 0.01.

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Mutagenesis, Expressing, Transformation Assay, Marker

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: NOTCH3 regulates PVR-mediated cell-cell interactions. (a) Total interaction number and strength comparison of NOTCH3-low and NOTCH3-high samples. (b) Overall interaction pattern of NOTCH3-low and NOTCH3-high samples. (c, d) Differential cell interactions (NOTCH3-high vs NOTCH3-low): (c) network diagrams and (d) heatmap. NOTCH3-low and NOTCH3-high specific pathways, displayed in (e) network and (f) heatmap. (g) Bubble plot showing the interaction pathways between cancer cells and immune cells (ILC, NK, CD4+ T, CD8+ T, γδ T and ZBTB16+ T cells), highlighting PVR pathway in NOTCH3-high samples.

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Comparison

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: NOTCH3 impairs CD8+ T cell cytotoxicity via PVR. (a) Heatmap of normalized enrichment score (NES) of immune-related KEGG pathways by comparing the transcriptome of NOTCH3-WT and NOTCH3-MT tumors, using GSEA. Red represents elevated and blue is decreased. Bladder Urothelial Carcinoma: 408, Brain Lower Grade Glioma: 515, Breast Invasive Carcinoma: 1083, Cervical Squamous Cell Carcinoma: 295, Colorectal Adenocarcinoma: 593, Esophageal Adenocarcinoma: 182, Glioblastoma Multiforme: 161, Head and Neck Squamous Cell Carcinoma: 516, Kidney Renal Clear Cell Carcinoma: 511, Liver Hepatocellular Carcinoma: 367, Lung Adenocarcinoma: 511, Lung Squamous Cell Carcinoma: 485, Mesothelioma: 88, Pancreatic Adenocarcinoma: 178, Prostate Adenocarcinoma: 494, Sarcoma: 254, Skin Cutaneous Melanoma: 444, Stomach Adenocarcinoma: 413, Uterine Corpus Endometrial Carcinoma: 528. (b) Immune-related pathway activation in NOTCH3-mutant CRC samples. (c) NOTCH3 and PVR expression was enhanced cancer samples compared to the normal in TCGA-CRC dataset. (d) PVR expression is correlated with NOTCH3 expression. (e) NOTCH3 mutated TCGA-CRC samples express lower PVR. (f) PVR expression was reduced in NOTCH3-mutant samples compared to NOTCH3-wild type. (g) PVR expression was reduced in NOTCH3 knocking down samples compared to the control. CD8+ T (primary cells isolated from whole blood) cytotoxicity assays: (h) LDH release and (i) cell viability (N.S p>0.05, ***p<0.001).

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Activation Assay, Mutagenesis, Expressing, Control, Isolation

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: NOTCH3-RBPJ axis transcriptionally activates PVR. (a) Venn diagram of PVR regulators intersecting NOTCH3 interacted proteins and transcription factors (TFs) of PVR. (b) Co-IP showing NOTCH3-RBPJ binding. (c) Nuclear NOTCH3 accumulation with WT vs. MT (subcellular fractionation). (d) Nuclear RBPJ accumulation with WT vs. MT (subcellular fractionation). (e) PVR promoter region has a RBPJ binding motif. (f) Luciferase reporter assay showing the activity of PVR promoter for RBPJ. (g) PVR induction by RBPJ overexpression (qPCR and Western blotting). (h) ChIP-PCR examing RBPJ binding to PVR promoter, using IgG and RBPJ antibody. (i) Synergistic PVR activation by NOTCH3+RBPJ. (n.s p>0.05, **p<0.01, ***p<0.001).

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Fractionation, Luciferase, Reporter Assay, Activity Assay, Over Expression, Western Blot, Activation Assay

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: NOTCH3 mutation enhances anti-PD-L1 response in vivo . (a) Processing steps of different grouped mice (N = 5 for each group). (b) NOTCH3, RBPJ and PVR expression in the IgG, anti-PD-L1 and anti-PD-L1+anti-NOTCH3 groups estimated by qRT-PCR. (c) Tumor volume overtime of different groups. (d) Tumor weight at sacrifice of different groups. (e) Cd8a abundance in three groups evaluated using Western blotting (n.s not significant, *p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Mutagenesis, In Vivo, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal cancer

doi: 10.3389/fimmu.2026.1741261

Figure Lengend Snippet: Clinical validation of NOTCH3 as an immunotherapy biomarker. (a) Survival difference between NOTCH3-low and NOTCH3-high pan-cancer patients who received immunotherapy (N = 954). (b) Survival difference between NOTCH3-wild-type and NOTCH3-mutant patients who received immunotherapy in MSKCC-CRC dataset (N = 109). (c) Multivariate Cox regression of for overall survival using NOTCH3 mutation and other clinical indicators in MSKCC dataset (N = 109). (d) NOTCH3 IHC in tumor vs. normal in the samples we collected (N = 102). (e) Clinical signatures associated with NOTCH3 expression. (f) Multivariate Cox regression of for overall survival using NOTCH3 mutation and other clinical indicators in our dataset (N = 102). (g) Kaplan-Meier analysis by NOTCH3 IHC levels (high vs. low; log-rank p<0.05, N = 102). (h) The mechanism of NOTCH3. n.s, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Mice were subcutaneously implanted with HCT116 CRC cells, followed by treatment with anti-PD-L1 antibody (10 mg/kg, administered every other day; Bio X Cell, USA) and/or NOTCH3 antibody (10mg/kg, 55114-1-AP, injected every the other day, proteintech, China) beginning two week post-injection.

Techniques: Biomarker Discovery, Mutagenesis, Expressing

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic representation of Notch signaling. (1) Furin (S1 cleavage) cleaves the NOTCH3 precursor protein in the Golgi system, resulting in a non-covalently bound heterodimeric protein that is transported to the cell surface. (2) A mechanical traction force is applied to the NOTCH3 ECD when a Notch ligand binds to the EGF repeats 10-11, exposing the extracellular NRR near the cell membrane, which consists of LNR and the heterodimerization domain (in green). Subsequently, ADAM17 cleaves the C-terminal portion of the heterodimerization domain (S2-cleavage). (3) The NEXT, which is made up of a RAM domain, many ANK domains, a PEST domain, and a transmembrane domain, is cleaved by the γ-secretase (S3-cleavage) releasing the N3ICD. (4) The N3ICD binds to the CSL complex protein and together with the co-activator Mastermind-like (MAM) trigger downstream gene transcription in the nucleus. (5) The NOTCH3 ECD and ligand are normally endocytosed by the ligand expressing cell and is degraded in the lysosome. B) Schematic representation of NOTCH3 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) mutations. NOTCH3 ECD contains 34 EGF repeat domains, each of which has 6 cysteine residues (WT). Mutations in CADASIL change the number of cysteines to an uneven number of cysteines (Mutant). These unpaired cysteines residues result in incorrect EGF repeat folding, irregular protein folding which leads to an enhanced NOTCH3 ECD multimerization. Distribution of the cysteine-altering mutations that cause CADASIL are shown. In the CADASIL mutant NOTCH3 ECD, the endocytosis is hampered, and NOTCH ECD remains outside of the VSMC and starts to accumulate and aggregate around the vessels. ADAM17, a disintegrin and metalloproteinase domain-containing protein 17; ANK, ankyrin repeats; EGF, epidermal growth factor; HD, heterodimerization domain; LNR, Lin-Notch repeats; PEST, proline (P), glutamic acid (E), serine (S) and threonine (T) degradation domain; RAM, Rbp-associated molecule domain; TM, transmembrane domain.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Membrane, Expressing, Mutagenesis

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic representation of NOTCH3 and NOTCH3 EGF 1-5 . NOTCH3 represents the full-length protein, and NOTCH3 EGF 1-5 represents the NOTCH3 protein with exon 1 to 5 fused with a myc-His-Tag at the C-terminus used for purification of the aggregated protein. B) Western blot of the NOTCH3 EGF 1-5 WT and R133C purified protein. The eluate fractions were visualized by western blot using an α-myc antibody. C) Western blot of NOTCH3 EGF 1-5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF 1-5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were verified after 1-5 days incubation by western blot using α-myc ab under non-reducing conditions.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Purification, Western Blot, Incubation

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic and work plan of the subcutaneous active immunization on the TgN3R182C 150 mouse model. B) Antibody titre validation of serum from TgN3R182C 150 CADASIL mice immunized with NOTCH3 EGF 1-5 aggregates (vaccinated) and PBS (sham) at 4, 5 and 7 months old. A direct ELISA with NOTCH3 aggregate-coated plates and different dilutions of serum was performed.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Biomarker Discovery, Direct ELISA

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF 1-5 - immunized mice at 7 months of age and TgN3R182C 150 at 18 months of age. Representative images show brain arteries of TgN3R182C 150 (7 and 18 months), sham and NOTCH3 EGF 1-5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α-SMA antibody (green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF 1-5 - immunized, sham and non-vaccinated TgN3R182C 150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C 150 mice at 18 months of age versus NOTCH3 EGF 1-5 - immunized, sham and non-vaccinated TgN3R182C 150 mice at 7 months of age. (*p < 0.05, **p < 0.01, ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: Quantitative real-time PCR analysis of the Notch downstream target genes NOTCH3, Hes1, Hey1 and Nrip2 on TgN3R182C150 mice at 5 and 12 months of age.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Real-time Polymerase Chain Reaction

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF 1-5 - immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C 150 , sham and NOTCH3 EGF 1-5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan antibody (green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3-ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3 months old TgN3R182C 150 (n=3) and 7 months old TgN3R182C 150 (n=6) mice and 18 months old TgN3R182C 150 (n=3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF 1-5 - immunized (n=11), sham (n=9) and non-vaccinated TgN3R182C 150 (n=6) mice. (*p < 0.05, **p < 0.01, ****p < 0.0001, ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: Quantification of human NOTCH3 ECD protein present in the whole blood serum of sham, immunized and non-vaccinated TgN3R182C 150 mice (at 3 and 7 months old). A) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C 150 mice at three months of age and further increased at seven months of age. B) NOTCH3 ECD in the TgN3R182C 150 mice was significantly reduced in the vaccinated TgN3R182C 150 mice. Statistical analysis was performed using unpaired t test with Welch’s correction. P < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Immunostaining for smooth muscle actin (ASMA) revealed that there were no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in WT (C57Bl6/J) versus TgN3R182C 150 mice at 7 months of age. B) Immunostaining for smooth muscle actin (ASMA) shows no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in NOTCH3 EGF 1-5 - vaccinated versus sham-vaccinated TgN3R182C 150 mice. C) Immunostaining for smooth muscle actin (ASMA) shows an extensive loss of VSMC in the Notch3 -/- mice when compared to a WT (C57Bl6/J) at 3 months of age. Scale bar =50µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Immunostaining

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF1-5 - immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1-5 - immunized (n=11), sham (n=9) and non-vaccinated TgN3R182C 150 (n=6) mice at 7 months of age. (ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

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

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: NIH3T3 cells were transfected with the control, wild type NOTCH3, or NOTCH3 R182C plasmids, as well as the β-gal and 12XCSL-luc reporter plasmids and cultured on immobilized jagged2 (Jag2) in the presence of DMSO or DAPT (n=3 and two technical replicates). Statistical analysis was performed using 2-way ANOVA followed by Tukey’s multiple comparisons tests. P < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ns= non-significant). RLU, relative luminescence units.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Transfection, Control, Cell Culture

Journal: Arthritis and rheumatism

Article Title: Hypoxia activates the notch signaling pathway in cells of the intervertebral disc: implications in degenerative disc disease.

doi: 10.1002/art.30246

Figure Lengend Snippet: Figure 1. Expression of Notch receptors and ligands, as well as Notch target gene Hes1, in rat intervertebral discs. A, Transverse sections of disc tissue from mature rats were treated with anti–Notch-4 antibodies, and expression of Notch-4 was detected in cells of the nucleus pulposus (NP) and annulus fibrosus (AF) (arrows). Original magnification 20. B, Expression of Notch-4, Jagged-1, and Hes1 was assessed in NP and AF tissue by Western blotting; anti–-tubulin was used as a positive control. C, Expression of select Notch ligands (Jagged1 [J1], Jagged2 [J2], Delta1, and Delta3) and Notch receptors (N1–N4) was assessed in AF tissue by reverse transcription–polymerase chain reaction (RT- PCR). D, Messenger RNA expression of the 4 Notch receptors was assessed in primary cells of the AF (left) and NP (right) by real-time RT-PCR. Values for Notch2–Notch4 are expressed relative to that of Notch1 and are the mean SEM results from 3 independent experiments. P 0.05. NS not significant. E, AF and NP cells were assessed by immunofluorescence analysis for the expression of Notch3, Notch4, and Jagged1, as well as cleaved Notch1 (Notch1– intracellular domain [N1-ICD]). Original magnification 20.

Article Snippet: After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, blocked with PBS containing 5% FBS, and incubated with antibodies against cleaved Notch1 (1:200; Cell Signaling Technology), Notch3 (1:200), Notch4 (1:100), or Hes1 (1:100) (the latter 3 from Neuromics), at 4°C overnight.

Techniques: Expressing, Western Blot, Positive Control, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Quantitative RT-PCR, Immunofluorescence

Journal: Arthritis and rheumatism

Article Title: Hypoxia activates the notch signaling pathway in cells of the intervertebral disc: implications in degenerative disc disease.

doi: 10.1002/art.30246

Figure Lengend Snippet: Figure 2. Hypoxic regulation of Notch receptor expression in rat disc cells. A–D, AF and NP cells were cultured under conditions of hypoxia (Hx) for 8 or 24 hours, or under normoxic (Nx) conditions, and the response to hypoxia was assessed by real-time reverse transcription– polymerase chain reaction analysis of mRNA expression for Notch receptors Notch1 (A), Notch2 (B), Notch3 (C), and Notch4 (D). Values are the mean SEM results from 3 independent experiments. P 0.05. E and F, Notch-4 protein expression under conditions of hypoxia, or under normoxic conditions, was assessed in AF and NP cells by immunofluorescence analysis (after 24 hours of hypoxia) (E) and Western blotting (after 8 and 24 hours of hypoxia) (F). Anti–- tubulin was used as a positive control in Western blots. Original magnification 20. See Figure 1 for other definitions.

Article Snippet: After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, blocked with PBS containing 5% FBS, and incubated with antibodies against cleaved Notch1 (1:200; Cell Signaling Technology), Notch3 (1:200), Notch4 (1:100), or Hes1 (1:100) (the latter 3 from Neuromics), at 4°C overnight.

Techniques: Expressing, Cell Culture, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence, Western Blot, Positive Control