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: 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