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: Journal of Biomedical Science

Article Title: A novel c-Kit/phospho-prohibitin axis enhances ovarian cancer stemness and chemoresistance via Notch3—PBX1 and β-catenin—ABCG2 signaling

doi: 10.1186/s12929-020-00638-x

Figure Lengend Snippet: Metastatic ovarian cancer cells enhance c-Kit, Notch3, and membrane raft-associated PHB and contribute to CSC enrichment. a Production of metastasized ovarian cancer cells from several passages of SKOV3GL cells into the mouse intraperitoneal cavity. The migration ability increased in SKOV3GL-G3 and SKOV3GL-G4 cells as determined by a 24-h Boyden chamber assay. b The levels of phospho-PHB Y259 , phospho-PHB T258 , and PHB in the lipid raft or cytosolic plus non-raft membrane (C + M) fraction of SKOV3GL, SKOV3GL-G1 to -G4 cells were detected by western blotting. c Expression of c-Kit, full-length (FL) Notch3 and membrane tethered Notch3 fragment (NTM) were analyzed from SKOV3GL to SKOV3GL-G4 cells by western blotting. d SKOV3GL-G4 cells were transfected with scrambled control or c-Kit siRNA for 48 h. Total cellular c-Kit, Notch3 (FL & NTM), phospho-PHB, PHB and β-actin protein expression was analyzed by western blotting. e Colony forming assay. SKOV3GL and SKOV3GL-G1 to -G4 cells were plated at a density of 300 cells/well in 6-well plates. f Phase contrast photomicrographs showing clonal expansion of SKOV3GL-G4 cells into the oncosphere over a 12-day period (top). Photomicrographs of SKOV3GL-G4 adherent cells, oncosphere expansion in low-adherence culture, and redifferentiated oncosphere cells after return to adherent culture (middle). qPCR for putative stem cell markers (Oct4, Nanog, SOX2) expressed as fold of SKOV3GL parental cells (bottom). Scale bar = 50 μm. All statistics represent the mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test. Blots are representative of three independent experiments. g SKOV3GL and SKOV3GL-G4 cells were seeded in CSC medium for 12 days. After 12 days, spheres were harvested and trypsinized into single cells. A total of 1000 or 100,000 cells in 50 μl DPBS were mixed with 50 μl Matrigel and subcutaneously injected into the flanks of nude mice. In vivo CSC properties were determined by luciferase signal at week 4 post injection (mean ± SD; n = 3 per group; **, P < 0.01; *, P < 0.05, t test)

Article Snippet: Cells were fixed with 4% paraformaldehyde and incubated with primary antibodies against Notch3 mouse monoclonal antibody (NBP2–52521, Novus Biologicals, Littleton, CO, USA) or c-Kit rabbit polyclonal antibody (3074, Cell Signaling Technology) and coincubated with 1 μM rhodamine-tagged CKGGRAKDC peptide (Genemed Synthesis, San Antonio, TX, USA) that stained for raft-PHB at 4 °C overnight.

Techniques: Membrane, Migration, Boyden Chamber Assay, Western Blot, Expressing, Transfection, Control, Injection, In Vivo, Luciferase

Journal: Journal of Biomedical Science

Article Title: A novel c-Kit/phospho-prohibitin axis enhances ovarian cancer stemness and chemoresistance via Notch3—PBX1 and β-catenin—ABCG2 signaling

doi: 10.1186/s12929-020-00638-x

Figure Lengend Snippet: c-Kit-mediated phospho-PHB Y259 in the membrane raft associates with Notch3 to regulate the Notch3—PBX1 signaling pathway. a Analysis of Notch3 (FL & NTM) and PBX1 protein expression in SKOV3_vector and SKOV3_c-Kit cells by western blotting. The quantification data represent the mean ± SD of three independent experiments. **, P < 0.01; *, P < 0.05, t test. b SKOV3 cells were transfected with empty vector, wild-type pD-PHB-HA , or mutant PHB plasmid ( pD-PHB Y259F -HA ) for 48 h. The levels of Notch3 (FL & NTM) and PBX1 were analyzed by western blotting. c Transfection with empty vector or mutant PHB plasmid ( pD-PHB Y259F -HA ) in SKOV3_c-Kit or SKOV3GL-G4 cells for 48 h. The levels of Notch3 (FL & NTM) and PBX1 were analyzed in whole cell lysate (WCL, top). Endo-PHB Y259 , PHB and exo-PHB expression was analyzed in the lipid raft domain (bottom) by western blotting. d SKOV3 cells transfected with mutant PHB plasmid ( pD-PHB Y259F -HA ) were treated with or without chloroquine (25 μM) (or MG132; 20 μM), and the level of Notch3 was determined by western blotting and compared to that in SKOV3 cells transfected with wild-type pD-PHB-HA plasmid. e To monitor protein stability, SKOV3 cells transfected with wild-type pD-PHB-HA or mutant PHB plasmid ( pD-PHB Y259F -HA ) were treated with cycloheximide (CHX; 50 μM), and the level of Notch3 was measured at the indicated time points. Data represent the mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test. f SKOV3_c-Kit cells were transiently transfected with plasmid pD-PHB-HA, and immunoprecipitation was performed with anti-HA tag antibody. Coimmunoprecipitation (Co-IP) proteins were detected by western blotting with anti-c-Kit, anti-PHB, and anti-Notch3 antibodies. g Localization of c-Kit, raft-PHB, and Notch3 was analyzed in SKOV3_c-Kit cells through confocal microscopy. Scale bar = 10 μm. h SKOV3 cells were transfected with empty vector, wild-type pD-PHB-HA , or mutant PHB plasmid ( pD-PHB Y259F -HA ) for 48 h. PHB was immunoprecipitated with anti-HA antibody, and coimmunoprecipitation of Notch3 was detected by western blotting. Blots are representative of three independent experiments

Article Snippet: Cells were fixed with 4% paraformaldehyde and incubated with primary antibodies against Notch3 mouse monoclonal antibody (NBP2–52521, Novus Biologicals, Littleton, CO, USA) or c-Kit rabbit polyclonal antibody (3074, Cell Signaling Technology) and coincubated with 1 μM rhodamine-tagged CKGGRAKDC peptide (Genemed Synthesis, San Antonio, TX, USA) that stained for raft-PHB at 4 °C overnight.

Techniques: Membrane, Expressing, Plasmid Preparation, Western Blot, Transfection, Mutagenesis, Immunoprecipitation, Co-Immunoprecipitation Assay, Confocal Microscopy