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: Oncology Reports

Article Title: Mangiferin induces apoptosis in human ovarian adenocarcinoma OVCAR3 cells via the regulation of Notch3

doi: 10.3892/or.2017.5814

Figure Lengend Snippet: Mangiferin regulates Notch3, Wnt and Akt pathway. (A) Western blot images of Notch3, cyclin D1, β-catenin, and PI3K/Akt/mTOR signaling pathway. (B) Overexpression of NICD (Notch3 intracellular domain). (C) Western blot images of cells treated with both mangiferin and NICD lentivirus. Images are representative from 3 independent experiments.

Article Snippet: Rabbit polyclonal pro-caspase-3 (cat. no. sc-7148; 1:1,000 dilution), mouse monoclonal pro-caspase-9 (cat. no. sc-56073; 1:1,000 dilution), rabbit polyclonal Bax (cat. no. sc-493; 1:500 dilution), mouse monoclonal Bid (cat. no. sc- 135847; 1:1,000 dilution), rabbit polyclonal Bcl-2 (cat. no. sc-492; 1:1,000 dilution), mouse monoclonal Bcl-X L (cat. no. sc-8392; 1:1,000 dilution), rabbit polyclonal PARP (cat. no. sc-7150; 1:1,000 dilution), rabbit polyclonal cytochrome c (cat. no. sc-7159; 1:1,000 dilution), mouse monoclonal cyclin D1 (cat. no. sc-450; 1:1,000 dilution), mouse monoclonal β-catenin (cat. no. sc-59737; 1:1,000 dilution), rabbit polyclonal Notch3 (NICD domain, cat. no. sc-5593; 1:1,000 dilution), mouse monoclonal Notch1 (cat. no. sc-376403; 1:1,000 dilution) and mouse monoclonal β-actin (cat. no. sc-47778; 1:5,000 dilution) purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Over Expression

Journal: Oncology Reports

Article Title: Mangiferin induces apoptosis in human ovarian adenocarcinoma OVCAR3 cells via the regulation of Notch3

doi: 10.3892/or.2017.5814

Figure Lengend Snippet: Notch3 overexpression abrogates the apoptosis-inducing effect of mangiferin. Representative phase contrast images (A) and fluorescent images (B) of OVCAR3 cells treated with mangiferin (left) and mangiferin plus NICD lentivirus (right). Images are ×200 magnifications with scale bar representing 100 µm. (C) Flow cytometry analysis of apoptosis by Annexin V and PI double staining. (D) Statistics of early apoptotic cells, late apoptotic cells and viable cells. *P<0.05 and **P<0.01 vs. pcDNA transfected OVCAR3 cells by Student's t-test. Data are means ± SEM from 3 independent experiments.

Article Snippet: Rabbit polyclonal pro-caspase-3 (cat. no. sc-7148; 1:1,000 dilution), mouse monoclonal pro-caspase-9 (cat. no. sc-56073; 1:1,000 dilution), rabbit polyclonal Bax (cat. no. sc-493; 1:500 dilution), mouse monoclonal Bid (cat. no. sc- 135847; 1:1,000 dilution), rabbit polyclonal Bcl-2 (cat. no. sc-492; 1:1,000 dilution), mouse monoclonal Bcl-X L (cat. no. sc-8392; 1:1,000 dilution), rabbit polyclonal PARP (cat. no. sc-7150; 1:1,000 dilution), rabbit polyclonal cytochrome c (cat. no. sc-7159; 1:1,000 dilution), mouse monoclonal cyclin D1 (cat. no. sc-450; 1:1,000 dilution), mouse monoclonal β-catenin (cat. no. sc-59737; 1:1,000 dilution), rabbit polyclonal Notch3 (NICD domain, cat. no. sc-5593; 1:1,000 dilution), mouse monoclonal Notch1 (cat. no. sc-376403; 1:1,000 dilution) and mouse monoclonal β-actin (cat. no. sc-47778; 1:5,000 dilution) purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Over Expression, Flow Cytometry, Double Staining, Transfection

Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 3 Expression of STAT5A is positively associated with Notch3 in breast cancer. a In TCGA (Cell 2015), the expression of STAT5A was positively associated with Notch3 in patient tissues. b Notch3 and STAT5A mRNA levels in different breast cancer cell lines. c STAT5A protein expression was positively associated with Notch3 in different breast cancer cell lines. d Overexpression of Notch3 promoted expression of STAT5A and its active form, p-STAT5, at the protein level in BT549 cells. e Overexpression of Notch3 promoted the expression of STAT5A and its active form, p-STAT5, at the protein level in MCF-7 cells. f In MCF-7 cells, knockdown of Notch3 decreased STAT5A and p-STAT5A at the protein level. g Overexpression of Notch3 increased the mRNA level of STAT5A in BT549 cells. h Overexpression of Notch3 increased STAT5A mRNA levels in MCF-7 cells. i The mRNA level of STAT5A was suppressed by siNotch3

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Expressing, Over Expression, Knockdown

Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 4 Notch3 directly binds to the STAT5A promoter and activates STAT5A expression. a Potential CSL binding sites of Notch3 on the STAT5A promoter, and the adjacent region, lacking Notch3 binding sites, as the negative control, and the construction strategy of luciferase reporter genes driven by wildtype and CSL-mutant STAT5A promoters. b ChIP results showing that Notch3 directly binds to the STAT5A-2/3 region on the promoter. c In MCF-7 cells, the luciferase activity of pGL3-STAT5A-pro-luc-E was suppressed by loss of Notch3 on the promoter region of STAT5A in a dose-dependent manner. d In MCF-7 cells, the luciferase activity of pGL3-STAT5A-pro-23-luc-E was suppressed by knockdown of Notch3 in a dose-dependent manner. The STAT5A promoter, lacking Notch3 binding sites, was not regulated by knockdown of Notch3. e In BT549 cells, STAT5A promoter-driven luciferase activity was activated by overexpression of Notch3 in a dose-dependent manner. f In BT549 cells, STAT5A promoter-driven luciferase activity was activated by the binding of Notch3 on the STAT5A promoter in a dose-dependent manner. The STAT5A promoter, lacking Notch3 binding sites, was not regulated by the overexpression of Notch3

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Expressing, Binding Assay, Negative Control, Luciferase, Mutagenesis, Activity Assay, Knockdown, Over Expression

Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 5 Notch3-mediated suppression of metastasis can be reversed by STAT5A siRNA in breast cancer cells and high Notch3 and STAT5A expression predicts better prognosis in patients with breast cancer. a Suppressive effect of Notch3 on wound healing was reversed by suppressing STAT5A expression in BT549 cells. b In transwell assays, Notch3-induced decreased mobility of BT549 cells was rescued by knockdown of STAT5A. c Enhancement of wound healing by siNotch3#1 was reversed by overexpression of STAT5A in MCF-7 cells. d Low expression of Notch3 predicted poor recurrence-free survival in patients with breast cancer. As well as, breast cancer patients with low STAT5A levels showed poor recurrence-free survival. e Low expression of Notch3 predicted poor overall survival in patients with breast cancer. And breast cancer patients with low STAT5A levels showed poor overall survival

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Expressing, Knockdown, Over Expression

Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 6 Proposed model of how Notch3 upregulate STAT5A in breast cancer. Notch3 is cleavaged twice to form N3ICD. In the cell nucleus, N3ICD forms a complex with CSL, which initiates transcription of STAT5A. Prolactin(Prl) binding to the receptor(PrlR) results in receptor dimerization and autophosphorylation of the receptor-associated JAK. Then, JAK phosphorylates the receptor, thereby creating docking sites for Src homology 2 (SH2) domain proteins, such as STAT5A. Subsequently, Jak2 phosphorylates the STAT5A to form an active dimer

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Binding Assay

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 1. Retinal lesions and ASCs are caused by Notch3 signaling. A, virus is microinjected into the ventricles of an E10.5 mouse embryo. At the time of injection, the forebrain and optic cup are a continuous structure, allowing viral infection of the anlage giving rise to the retina and optic nerve. Viral particles are released into the amniotic sac near the injection site on needle withdrawal and infect the developing lens. B, E17.5 retina in a N3CLE-injected animal with a PLAP-positive clone (i). H&E staining showed that N3CLE-induced retinal lesions can span 1 (ii) or all 3 layers (iii) of the retina. Retinal lesions contained BIII- tubulin–positive (iv) and GS-positive (v) cells that were often double positive for nuclear Notch3 (N3). C, Pax6/Chx10 double-positive cells in retinal lesions (arrowheads). D, i, white cataractous lenses developed in N3CLE-injected animals. ii, the ALE (arrowhead) expanded into multiple epithelial layers (arrow) and contained mitotic bodies in the N3CLE-induced ASCs (inset). iii, areas of hyperproliferation were PLAP positive and contained Ki67-positive cells (DAPI counterstain). Original magnifications: 20 (Bi, Biv, and C), 40 (Bv and Dii), 200 (Bii, Biii, and Di).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Virus, Injection, Infection, Staining

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 2. Invasive choroidal tumors and retinal glial lesions arise following Notch3 activation. N3CLE-induced cellular choroidal tumors in the adult eye were PLAP positive (A), pigmented, and arose in the choroidal layer beneath an intact retinal pigment epithelium (Bi, arrow). Many choroidal tumors invaded outward through breaks in the sclera (Bi, arrowheads) and could diffusely infiltrate periocular tissues. Choroidal tumors contained nuclear N3 and Ki67-positive cells (Bii and Biii). C and D, N3CLE-injected animals contained PLAP-positive retinal glial lesions (C), GFAP-positive (Dii), and GS-positive (Diii). Original magnifications: 10 (A), 20 (Bi), 40 (Bii and Diii), 63 (Biii, Di, and Dii), 64 (Bii), 200 (C).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Activation Assay, Injection

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 3. N3CLE induced formation of glial tumors along the optic nerve. A, glial tumors (arrowheads) formed along the optic nerve (arrow) in N3CLE-injected animals and contained mitotic bodies (ii, inset). B, the optic nerve tumors expressed GFAP and Notch3 and invaded orbital tissues surrounding the optic nerve. Some glial tumors involved both the retina (arrowhead) and optic nerve (arrow). C, i, glial tumors contained Ki67-positive proliferating cells. Nestin-positive and Nestin/GFAP-positive cells within the lesions coexpressing Hes5 or Notch3, respectively (Cii and D). Original magnifications: 63 (C and D), 100 (Ai and Bi), 200 (Ai, Aii, and Bii), 400 (Aii, inset).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Injection

Journal: Developmental cell

Article Title: Spatial-temporal lineage restrictions of embryonic p63 + progenitors establish distinct stem cell pools in adult airways

doi: 10.1016/j.devcel.2018.03.001

Figure Lengend Snippet: Key resources table

Article Snippet: rabbit anti-Notch3 , CST , Cat#5276; RRID:AB_10560515.

Techniques: Recombinant, Imaging