Journal: Brain : a journal of neurology

Article Title: Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

doi: 10.1093/brain/aww011

Figure Lengend Snippet: Figure 1 Schematic representation of wild-type and mutant NOTCH3. (A) The ectodomain of the wild-type NOTCH3 protein contains 34 EGFr domains, each with six cysteine residues forming three disulphide bridges between specific pairs of cysteine residues. (B) In mutant NOTCH3, there is an unpaired cysteine residue in the EGFr domain affected by the mutation. The example shown in this figure is the common p.Arg153Cys mutation in exon 4 (indicated in red). As a result of the mutation, EGFr 3 contains seven cysteine residues, resulting in an unpaired cysteine and disrupted disulphide bridge formation.

Article Snippet: Cells were blocked with 1% bovine serum albumin (BSA) in PBS for 20 min and were incubated overnight at 4 C with an antibody against the extracellular part of NOTCH3 (anti-NOTCH3ECD, Novus Biologicals; dilution 1:5000).

Techniques: Mutagenesis, Residue

Journal: Brain : a journal of neurology

Article Title: Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

doi: 10.1093/brain/aww011

Figure Lengend Snippet: Figure 2 NOTCH3 cysteine correction using exon skipping. (A) Schematic overview of the open reading frame of exons 2–24 of NOTCH3. The vertical boxes indicate the number of reported distinct CADASIL-causing mutations in each exon (Rutten et al., 2014). All CADASIL-causing mutations are located in exons 2–24, the exons that encode the EGFr domains of NOTCH3. The nine exon skip strategies, which are predicted to accomplish NOTCH3 cysteine correction with correct cysteine spacing, including a total of 12 exons skipped singly or in pairs, are underlined in green. (B and C) NOTCH3 cysteine correction for exon 4–5 skipping, as the seminal model for this approach. (B) Exons 4 and 5 together encode part of EGFr 2, all of EGFr 3, 4 and 5, and part of EGFr 6. Pre-mRNA NOTCH3 exon 4–5 skipping using AONs thus leads to the exclusion of the mutated EGFr 3 (shown in red) from the protein, as well as the complete exclusion of EGFr domains 4 and 5, and a partial exclusion of EGFr domains 2 and 6. The remaining amino acids of EGFr domains 2 and 6 (encoded by exons 3 and 6, respectively), form an EGFr 2–6 fusion domain with six cysteine residues that are correctly spaced within the domain. The fusion site of this domain is located between the fifth cysteine derived from EGFr 2 and the sixth cysteine derived from EGFr 6. The number of amino acids between the fifth and sixth cysteine is exactly according to wild-type, namely eight. (C) 3D homology modelling of the EGFr 2–6 fusion domain predicts a stable conformation and normal disulphide bridge formation. Disulphide bridges formed between cysteines are shown in yellow. The unpaired cysteine originating from the Arg153Cys mutation is shown in red. The disulphide bridge formed between the fifth and sixth cysteine in the EGFr 2–6 fusion domain is shown in orange.

Article Snippet: Cells were blocked with 1% bovine serum albumin (BSA) in PBS for 20 min and were incubated overnight at 4 C with an antibody against the extracellular part of NOTCH3 (anti-NOTCH3ECD, Novus Biologicals; dilution 1:5000).

Techniques: Derivative Assay, Mutagenesis

Journal: Brain : a journal of neurology

Article Title: Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

doi: 10.1093/brain/aww011

Figure Lengend Snippet: Figure 3 Expression and cell surface localization of NOTCH3 skip proteins. (A)Western blot analysis demonstrating expression of NOTCH3 skip proteins, after transient transfection of NOTCH3 cDNA constructs in HEK293 cells. NOTCH3 detection with an antibody against the NOTCH3 intracellular domain reveals the presence of the unprocessed full-length protein (FL), as well as the intracellular domain (IC). The full-length skip proteins run slightly lower on the gel than the wild-type NOTCH3, as a result of the small size difference. (B) Immunocytochemical analysis of fibroblasts transfected with the same NOTCH3 cDNA constructs. Staining of permeabilized cells with an antibody directed against the extracellular part of the protein shows intracellular NOTCH3 staining, which is predominantly perinuclear and is comparable between the wild- type NOTCH3 and NOTCH3 skip proteins. Staining of unpermeabilized cells reveals normal cell surface localization of NOTCH3 skip proteins.

Article Snippet: Cells were blocked with 1% bovine serum albumin (BSA) in PBS for 20 min and were incubated overnight at 4 C with an antibody against the extracellular part of NOTCH3 (anti-NOTCH3ECD, Novus Biologicals; dilution 1:5000).

Techniques: Expressing, Western Blot, Transfection, Construct, Staining

Journal: Brain : a journal of neurology

Article Title: Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

doi: 10.1093/brain/aww011

Figure Lengend Snippet: Figure 4 Ligand binding and ligand-induced signalling of NOTCH3 skip proteins. (A) Fibroblasts were transfected with NOTCH3 cDNA and incubated with a jagged 1-Fc peptide to allow for ligand binding. The jagged 1-Fc peptide was preclustered to an anti-Fc antibody (red fluorescence). The NOTCH3 expressing cells are visualized using an anti NOTCH3ECD antibody (green fluores- cence). Cells transfected with wild-type NOTCH3 cDNA show an increased jagged 1 staining where the NOTCH3 protein is ex- pressed, whereas this staining is absent in the cells transfected with NOTCH3 lacking the ligand binding domain (del EGFr 10–11). The cells expressing the NOTCH3 skip proteins all show an increased jagged 1 staining similar to wild-type NOTCH3, indicating normal ligand binding. Images are shown from one representative experi- ment at a 40 magnification. (B) NIH3T3 cells transiently trans- fected with NOTCH3 cDNA, were co-cultured with jagged 1 expressing cells to induce NOTCH3 signalling, and assayed for luciferase activity using a hexamerized CBF1 reporter plasmid. Cells transfected with wild-type NOTCH3 cDNA show a significant in- crease in luciferase activity upon co-culture with 3T3 cells ex- pressing human jagged 1, compared to co-culture with mock transfected 3T3 cells not expressing human jagged 1. The cells transfected with the respective skip NOTCH3 cDNA also showed a significant increase in luciferase activity. There was no significant

Article Snippet: Cells were blocked with 1% bovine serum albumin (BSA) in PBS for 20 min and were incubated overnight at 4 C with an antibody against the extracellular part of NOTCH3 (anti-NOTCH3ECD, Novus Biologicals; dilution 1:5000).

Techniques: Ligand Binding Assay, Transfection, Incubation, Expressing, Staining, Cell Culture, Luciferase, Activity Assay, Plasmid Preparation, Co-Culture Assay

Journal: Brain : a journal of neurology

Article Title: Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

doi: 10.1093/brain/aww011

Figure Lengend Snippet: Figure 5 Successful AON-induced NOTCH3 exon skipping in VSMCArg153Cys. RT-PCR and sequencing analysis of RNA from VSMCArg153Cys transfected with NOTCH3 AONs or control AON. Depicted are results from (A) exon 4–5 skipping, (B) exon 2–3 skipping and (C) exon 6 skipping. The RT-PCR of the samples transfected with the NOTCH3 AONs show that the intensity of the wild-type band is decreased and an additional, smaller product is present, which corresponds to the expected fragment length after exon skipping. Sequencing analysis of these smaller fragments confirmed that all three exon skips were successful. For the exon 4–5 skip, some isolated exon 5 skipping was seen in addition to joint exon 4–5 skipping.

Article Snippet: Cells were blocked with 1% bovine serum albumin (BSA) in PBS for 20 min and were incubated overnight at 4 C with an antibody against the extracellular part of NOTCH3 (anti-NOTCH3ECD, Novus Biologicals; dilution 1:5000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Control, Isolation

Journal: Brain : a journal of neurology

Article Title: Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

doi: 10.1093/brain/aww011

Figure Lengend Snippet: Figure 6 Expression of NOTCH3 and NOTCH3 target genes after exon skipping. (A) NOTCH3 quantitative PCR ana- lysis of VSMCcontrol transfected with NOTCH3 AONs. After exon skipping, total NOTCH3 expression levels are unaltered, whereas transfection with a downregulating NOTCH3 gapmer AON results in decreased NOTCH3 expression levels (unpaired Student’s t-test, P = 0.04). (B) Quantitative PCR analysis of NOTCH3 downstream target gene expression in VSMCcontrol transfected with NOTCH3 AONs. Transfection with NOTCH3 exon skipping AONs did not affect the expression levels of the canonical NOTCH3 downstream target genes HES1 and HEYL, as opposed to transfection with downregulating NOTCH3 gapmer AONs, which did lead to reduced expression of NOTCH3 target genes (unpaired Student’s t-test, HES1 P = 0.009, HEYL P = 0.09). Data represent the mean standard deviation from three independent experiments. ns = not significant.

Article Snippet: Cells were blocked with 1% bovine serum albumin (BSA) in PBS for 20 min and were incubated overnight at 4 C with an antibody against the extracellular part of NOTCH3 (anti-NOTCH3ECD, Novus Biologicals; dilution 1:5000).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Lysis, Transfection, Targeted Gene Expression, Standard Deviation

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: Methods (San Diego, Calif.)

Article Title: Generation of anti-Notch antibodies and their application in blocking Notch signalling in neural stem cells

doi: 10.1016/j.ymeth.2012.07.008

Figure Lengend Snippet: Receptor specific and dose dependent inhibition of Notch signalling between cells. Luciferase based co-culture assays were conducted with cells expressing the ligand Jagged1 and cells expressing different full-length Notch receptors (HEK-Notch1, HEK-Notch2 and HEK-Notch3). (A) Samples tested were anti-Notch1 antibody N1_E6, anti-Notch2 antibodies N2_B6 and N2_B9, the anti-human Notch3 antibody N3_mAb. Controls are non-activated, Jag1(-), and untreated, Jag1(+), cultures. The ratio of Firefly/Renilla luciferase activity is expressed relative to that of a control antibody (Shc1). (B) Dose response curves were generated by titration of N1_E6 on HEK-Notch1 and antibodies N2_B6 and N2_B9 on HEK-Notch2. The relative luciferase activity in non-activated cells were used to reflect 100% inhibition and the standard deviations were calculated to be 100 ± 6,65% for HEK-Notch1 (D) and 100 ± 2,51% for HEK-Notch2.

Article Snippet: Along with the blocking antibodies, an anti-Notch3 antibody, N3(E10), previously selected as a scFv antibody against murine Notch3 (R&D systems) (unpublished), was also converted to scFv-Fc for use in flow cytometry ( ).

Techniques: Inhibition, Luciferase, Co-Culture Assay, Expressing, Activity Assay, Control, Generated, Titration

Journal: Methods (San Diego, Calif.)

Article Title: Generation of anti-Notch antibodies and their application in blocking Notch signalling in neural stem cells

doi: 10.1016/j.ymeth.2012.07.008

Figure Lengend Snippet: Antibody-mediated inhibition of endogenous mouse and human Notch receptor signalling in neural stem cells. qRT-PCR was used to analyse the relative mRNA levels of Notch pathway genes in neural stem cell systems of both mouse and human origin. (A) Mouse NS cells express several Notch receptors and ligands. (B) Anti-Notch antibodies caused down-regulation of Notch dependent Hes5 expression in mouse NS cells relative to control antibody (Control ab). Inhibition of Hes5 with anti-NRR1 (N1) and anti-NRR2 (N2) antibodies are additive and co-incubation reduces Hes5 to the same extent as DAPT. (C) Notch receptors 1–3 and the ligands JAGGED1 and DLL1 and DLL3 are expressed in human NES cells (AF22). (D) Treating human NES cells with blocking antibodies targeting NRR1 (N1), NRR2 (N2) or NRR3 (N3) reduces HES5 expression. Co-incubation of anti-Notch1 with either anti-Notch2 or anti-Notch3 antibodies reduces HES5 expression to the same extent as inhibition with DAPT. Bars show relative mRNA levels as values normalised to GAPDH based on two separate reactions (A and C). Bars in figure B and D show fold change compared to DMSO control experiments.

Article Snippet: Along with the blocking antibodies, an anti-Notch3 antibody, N3(E10), previously selected as a scFv antibody against murine Notch3 (R&D systems) (unpublished), was also converted to scFv-Fc for use in flow cytometry ( ).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Control, Incubation, Blocking Assay