Journal: Oncogene

Article Title: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

doi: 10.1038/onc.2016.5

Figure Lengend Snippet: Pin1 silencing modulates the Notch3 protein expression in human T-ALL cell lines. Activated Notch1 (Notch1 Val1744 ) and Notch3 (N3 IC ) expression in response to Pin1 silencing in ( b , c ) Notch1-activated (Molt3, SilAll, P12-Ichikawa and Jurkat) and ( e – g ) Notch1-non activated/Notch3 activated (N3 IC-act ) overexpressing (TALL-1) human T-ALL cell lines. ( a , d ) Western blots against Pin1 show the efficiency of Pin1 silencing (siPin1) (left panels). Western blot against the anti-β-actin was used as a loading control. All the western blots in the figure are representative of at least three independent experiments, each in triplicate. In all right ( a – d ) and lower ( f , g ) panels are shown the optical densitometry (OD) of Pin1 ( a , d ), Notch1 ( b ) and Notch3 ( c , f , g ) protein expression levels analyzed in all the experiments performed, thus including the P -values, calculated using Student's T -test (i.e., ns, not significant P> 0.05; * P ⩽0.05; ** P ⩽0.01).

Article Snippet: Cells were treated with 10 μ M of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Cat#565770) for 24 h. In some cases, TALL-1 cells were treated with: 30 μ M proteasome inhibitor MG132 (Sigma, St Louis, MO, USA; Cat#C2211); 10 μg/ml ribosome inhibitor cycloheximide (Sigma; Cat#C4859) for the times indicated; 10 μg/ml of blocking anti-human Notch3 antibody (R&D Systems, Minneapolis, MN, USA; Cat#AF1559) for 48 h. Purified Sheep IgG (R&D Systems; Cat#5-001-A) was used as an isotype control.

Techniques: Expressing, Western Blot, Control

Journal: Oncogene

Article Title: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

doi: 10.1038/onc.2016.5

Figure Lengend Snippet: Pin1 silencing influences the TALL-1 cells invasiveness by regulating N3 IC protein expression. ( a ) Western blots against Pin1 show the efficiency of Pin1 silencing in TALL-1 cell line (siPin1). ( b ) TALL-1 cell line silenced or not for Pin1 was used in invasion Matrigel assay: relative percentage of invasiveness is shown with respect to the negative control, siCTR (left panel). RT–PCRs show downmodulation of MMP9 mRNA expression in Pin1-silenced cells (siPin1) with respect to the control cells (siCTR) (right panel). ( c ) Western blots against activated-N3 IC protein (N3 IC-act ) and Pin1 show the efficiency of the Notch3 receptor block and Pin1 silencing, respectively (lower panels). Optical densitometry (OD) of the activated-N3 IC protein expression (upper panel). ( d ) RT–PCRs show downmodulation of MMP9 mRNA expression in Notch3-blocked Pin1-silenced cells (siPin1+FCNotch3) with respect to both Notch3-blocked or Pin1-silenced controls alone. In both panels ( a ) and ( c ), western blot against the anti-β-actin was used as a loading control. All the results shown in the figure are expressed as the means average deviations of three separate experiments, each in triplicate, and P -values were calculated using Student's T -test (i.e., ns, not significant P> 0.05; * P ⩽0.05; ** P ⩽0.01). WCEs, whole-cell extracts.

Article Snippet: Cells were treated with 10 μ M of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Cat#565770) for 24 h. In some cases, TALL-1 cells were treated with: 30 μ M proteasome inhibitor MG132 (Sigma, St Louis, MO, USA; Cat#C2211); 10 μg/ml ribosome inhibitor cycloheximide (Sigma; Cat#C4859) for the times indicated; 10 μg/ml of blocking anti-human Notch3 antibody (R&D Systems, Minneapolis, MN, USA; Cat#AF1559) for 48 h. Purified Sheep IgG (R&D Systems; Cat#5-001-A) was used as an isotype control.

Techniques: Expressing, Western Blot, Matrigel Assay, Negative Control, Control, Blocking Assay

Journal: Oncogene

Article Title: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

doi: 10.1038/onc.2016.5

Figure Lengend Snippet: Pin1 ablation impairs Notch3 signaling in thymocytes of young N3 IC transgenic mice resulting in the decrease of expansion/invasiveness of CD4 + CD8 + DP splenic cells. CD4 + and/or CD8 + subset distribution of thymocytes from representative 6-week-old Pin1 +/+ (A), N3 IC -tg (B) and N3 IC -tg/Pin1 −/− (C) mice. ( b ) Whole-cell extracts from thymocytes illustrated in ( a ) were revealed with anti-Pin1, anti-activated N3 IC (N3 IC-act ), anti-HA (left panels) and anti-activated Notch1 (Notch1 Val1744 ), anti-Hes1 and anti-pTα (right panels) antibodies. Western blot against the anti-β-actin was used as a loading control. ( c ) CD4 + and/or CD8 + subset distribution of lymphocytes derived from SPL and blood of representative 6-week-old Pin1 +/+ (D), N3 IC -tg (E) and N3 IC -tg/Pin1 −/− (F) mice. ( d ) Sorted CD4 + CD8 + (DP) splenocytes illustrated in ( c ) (circle around the number) were used for western blot analysis against anti-activated N3 IC (N3 IC-act ), anti-HA and anti-β-actin antibodies and ( e ) in invasion Matrigel assay: relative percentage of DP cells invasiveness from N3 IC -tg/Pin1 −/− mice is shown with respect to N3 IC -tg cells. Results are shown as the means average deviations of five independent experiments ( n= 3–5 mice per group) and P -values were calculated using Student's T -test (i.e., ** P ⩽0.01). In all panels described in ( a , c ), numbers inside each cytogram indicate the percentages of the corresponding subsets and the results are representative of five independent experiments ( n= 3–5 mice per group: Pin1 +/+ ( n= 15), N3IC-tg ( n= 25) and N3IC-tg/Pin1 −/− mice ( n= 15)). THY, thymus. SPL, Spleen; PB, Peripheral Blood.

Article Snippet: Cells were treated with 10 μ M of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Cat#565770) for 24 h. In some cases, TALL-1 cells were treated with: 30 μ M proteasome inhibitor MG132 (Sigma, St Louis, MO, USA; Cat#C2211); 10 μg/ml ribosome inhibitor cycloheximide (Sigma; Cat#C4859) for the times indicated; 10 μg/ml of blocking anti-human Notch3 antibody (R&D Systems, Minneapolis, MN, USA; Cat#AF1559) for 48 h. Purified Sheep IgG (R&D Systems; Cat#5-001-A) was used as an isotype control.

Techniques: Transgenic Assay, Western Blot, Control, Derivative Assay, Matrigel Assay

Journal: Oncogene

Article Title: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

doi: 10.1038/onc.2016.5

Figure Lengend Snippet: Pin1 directly interacts with Notch3. ( a ) Control or anti-Flag antibody immunoprecipitates from HEK293T cells transfected with Flag N3IC-wt were subjected to far western blotting using purified GST–Pin1 as a probe, followed by anti-Pin1 immunoblotting. Anti-Flag western blot analysis of the upper panel after stripping is shown. ( b ) Lysates used in ( a ), previous treated with lamba phosphatase (+), were subjected to GST or GST–Pin1 pulldown followed by anti-Flag western blotting. The arrows indicate the phosphorylated (upper band) and the non-phosphorylated (lower band) forms. ( c ) Control or anti-Flag antibody immunoprecipitates from HEK293T cells co-transfected with Flag N3IC-wt and HA-Pin1 plasmids were subjected to western blot and probes with anti-MPM-2, to detect the Notch3 phosphorylation levels at Ser/Thr-Pro sites, followed by stripping and anti-Flag western analysis to show N3 IC immunoprecipitated protein levels. The blot with anti-HA antibody was used to reveal the Notch3-Pin1 binding (middle panel). The * indicates a non-specific band. ( d ) Control or anti-Pin1 antibody immunoprecipitates from the same cells used in ( c ) were probes with anti-Flag, to detect the Notch3-Pin1 binding, and with the anti-HA antibody to show Pin1 immunoprecipitated protein levels. ( e ) Anti-Notch3 (left panel) and anti-Pin1 (right panel) immunoprecipitates from N3–232 T cells were subjected to western blot and probes with anti-MPM2 antibody, to detect the Notch3 phosphorylation levels at Ser/Thr-Pro sites, and anti-N3 IC antibody to detect endogenous Notch3–Pin1 interaction, respectively. In both panels ( e ), the blots with anti-N3 IC and anti-Pin1 antibodies were used to show Notch3 and Pin1 immunoprecipitated protein levels, respectively. ( f ) Anti-Pin1 immunoprecipitates from N3IC-tg thymocytes were subjected to western blot and probes with anti-N3 IC and anti-Pin1 antibodies, to detect endogenous Notch3–Pin1 interaction and Pin1 immunoprecipitated protein levels, respectively. The input lane indicated in all the western blot of ( a – d ) shows 5% of total lysate. All data are representative of at least three independent experiments, each in triplicate. WCEs, whole-cell extracts.

Article Snippet: Cells were treated with 10 μ M of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Cat#565770) for 24 h. In some cases, TALL-1 cells were treated with: 30 μ M proteasome inhibitor MG132 (Sigma, St Louis, MO, USA; Cat#C2211); 10 μg/ml ribosome inhibitor cycloheximide (Sigma; Cat#C4859) for the times indicated; 10 μg/ml of blocking anti-human Notch3 antibody (R&D Systems, Minneapolis, MN, USA; Cat#AF1559) for 48 h. Purified Sheep IgG (R&D Systems; Cat#5-001-A) was used as an isotype control.

Techniques: Control, Transfection, Far Western Blot, Purification, Western Blot, Stripping Membranes, Phospho-proteomics, Immunoprecipitation, Binding Assay

Journal: Oncogene

Article Title: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

doi: 10.1038/onc.2016.5

Figure Lengend Snippet: Pin1 affects Notch3 processing. ( a ) CD4 + and/or CD8 + subset distribution of thymocytes from Pin1 +/+ and Pin1 −/− mice. In both panels, numbers inside each cytogram indicate the percentages of the corresponding subsets. ( b ) RT–PCR shows the unchanged relative Notch3 mRNA levels in Pin1 −/− vs Pin1 +/+ thymocytes (left panel). (Right panel) Western blot analysis of whole-cell extracts (WCEs) from the same thymocytes probed with anti-Notch3EC (N3 EC ) and anti-Pin1 antibodies. The β-actin expression was used as a loading control. ( c ) Notch3 extracellular expression (N3 EC ) from thymocytes of Pin1 +/+ and Pin1 −/− mice indicated as percentages inside each cytogram. The violet curve represents the isotypic control. The mean fluorescence intensity (MFI) ratio between Notch3 and isotypic control staining is also indicated. The results showed in both panels are representative of five independent experiments ( n= 5 mice for group). ( d ) Bar graphs represent the absolute cell number from thymocytes expressing N3 EC of the same mice indicated in ( c ). ( e ) Cytosolic (C) and membrane (M) fractions from Pin1 +/+ and Pin1 −/− thymocytes were analyzed in immunoblot assays to detect the N3 EC expression. Anti-Lck and anti-α-tubulin were used as fraction markers; anti-β-actin was used as a loading control. ( f ) Thymocytes from Pin1 +/+ and Pin1 −/− mice were incubated with EZ-Link Sulfo-NHS-SS-Biotin (+) or were mock (−) treated, as described in Materials and methods. Cells were lysed and extracts were loaded on a 6% SDS–PAGE gel either directly (T fraction, 15% of the extract) or after incubation on streptavidin-agarose beads (B fraction, 85% of the extract). Extracts were then immunoblotted with the anti-N3 EC and anti-N3 IC antibodies. Positions of the 210-kDa Notch3 extracellular (EC) and 97-kDa Notch3 transmembrane-intracellular (TM-IC) domains are indicated by black arrows. In the high exposition is indicated the position of the Notch3 intracellular domain (IC) (red arrow). ^ indicates non-specific bands. ( g ) Nuclear fractions from Pin1 +/+ and Pin1 −/− thymocytes were analyzed in immunoblot assays to detect the N3 IC expression. Anti-LaminB and anti-α-tubulin were used as fraction markers; anti-β-actin was used as a loading control. In all panels ( b ) and ( d ), results are shown as the means average deviations of five separate experiments and P -values were calculated using Student's T -test (i.e., ns, not significant P> 0.05; ** P ⩽0.01). In all the western blots represented in the figure, FL indicates Notch3 full-length receptor and EC indicates extracellular region.

Article Snippet: Cells were treated with 10 μ M of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Cat#565770) for 24 h. In some cases, TALL-1 cells were treated with: 30 μ M proteasome inhibitor MG132 (Sigma, St Louis, MO, USA; Cat#C2211); 10 μg/ml ribosome inhibitor cycloheximide (Sigma; Cat#C4859) for the times indicated; 10 μg/ml of blocking anti-human Notch3 antibody (R&D Systems, Minneapolis, MN, USA; Cat#AF1559) for 48 h. Purified Sheep IgG (R&D Systems; Cat#5-001-A) was used as an isotype control.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Fluorescence, Staining, Membrane, Incubation, SDS Page

Journal: Oncogene

Article Title: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

doi: 10.1038/onc.2016.5

Figure Lengend Snippet: Pin1 influences Notch3 processing and stability in endogenous and exogenous system. ( a ) Western blot analysis of Notch3 extracellular (N3 EC ) and activated intracellular (N3 IC-act ) protein expression of whole-cell extract (WCE) derived from Pin1-silenced TALL-1 (+) vs control cells (−) (left panel). The western blots in the figure are representative of at least three independent experiments, each in triplicate. The optical densitometry (OD) (right panels) was analyzed in all the experiments performed, thus including the P -values, calculated using Student's T -test (i.e., ** P ⩽0.01). ( b ) WCEs from Pin1-silenced TALL-1 cells (+) vs control cells (−) in a time course assay with 10 μg/ml of cycloheximide (CHX), in the presence or absence of the proteasome inhibitor MG132 for the same times before lysis, were revealed by immunoblotting with anti-activated N3 IC (N3 IC-act ), anti-Pin1 and anti-β-actin antibodies (left panel). The right panel shows the relative quantification of activated-N3 IC as determined by OD. ( c ) Left panel, Western blot analysis of whole-cell extracts from HEK293T cells transfected with Flag N3IC-wt plasmid and silenced for Pin1 (+) or control (−) in a time course assay with 10 μg/ml of cycloheximide (CHX). Extracts were immunoblotted with anti-Flag, anti-Pin1 and anti-β-actin antibodies. The right panel shows the relative quantification of Flag N3IC as determined by OD. All data are representative of at least three independent experiments, each in triplicate.

Article Snippet: Cells were treated with 10 μ M of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Cat#565770) for 24 h. In some cases, TALL-1 cells were treated with: 30 μ M proteasome inhibitor MG132 (Sigma, St Louis, MO, USA; Cat#C2211); 10 μg/ml ribosome inhibitor cycloheximide (Sigma; Cat#C4859) for the times indicated; 10 μg/ml of blocking anti-human Notch3 antibody (R&D Systems, Minneapolis, MN, USA; Cat#AF1559) for 48 h. Purified Sheep IgG (R&D Systems; Cat#5-001-A) was used as an isotype control.

Techniques: Western Blot, Expressing, Derivative Assay, Control, Lysis, Quantitative Proteomics, Transfection, Plasmid Preparation

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: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Schematic representation of Notch signaling. (1) Furin (S1 cleavage) cleaves the NOTCH3 precursor protein in the Golgi system, resulting in a non‐covalently bound bipartite 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, the 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 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 degraded in the lysosome. 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 six 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: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Membrane, Expressing, Mutagenesis

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: 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 1–5 fused with a myc‐His‐Tag at the C‐terminus used for purification of the aggregated protein. Western blot of the NOTCH3 EGF 1–5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α‐myc antibody. 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 visualized after 1–5 days incubation by western blot using α‐myc antibody under non‐reducing conditions. Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Purification, Western Blot, Incubation

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Schematic pipeline of the subcutaneous active immunization in the TgN3R182C150 mouse model. Antibody titer validation of serum from TgN3R182C150 CADASIL mice immunized with NOTCH3 EGF 1–5 aggregates (vaccinated, n = 11) and PBS (sham, n = 9) at 4, 5, and 7 months old. A direct ELISA with NOTCH3 aggregate‐coated plates and different dilutions of serum was performed. Error bars indicate standard error of the mean (SEM).

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Biomarker Discovery, Direct ELISA

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐immunized mice at 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (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). Scale bar = 20 μm. 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 ( n = 10), sham ( n = 8), and non‐vaccinated ( n = 6) TgN3R182C150 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 TgN3R182C150 mice at 18 months ( n = 3) of age versus NOTCH3 EGF 1–5 ‐immunized ( n = 10), sham ( n = 8), and non‐vaccinated ( n = 6) TgN3R182C150 mice at 7 months of age. Statistical significance was assessed using a Brown‐Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons (NOTCH3 ECD deposits (% of vessel area): 7 m.o. vs. Sham, ns P = 0.9424; 7 m.o. vs. Vaccinated, ns P = 0.9809; 7 m.o. vs. 18 m.o. ** P = 0.0046; Sham vs. Vaccinated, ns P = 0.9999; Sham vs. 18 m.o. ** P = 0.0032; Vaccinated vs. 18 m.o. ** P = 0.0032. NOTCH3 ECD deposits (number/1,000 μm 2 ): 7 m.o. vs. Sham, ns P = 0.7877; 7 m.o. vs. Vaccinated, ns P = 0.8358; 7 m.o. vs. 18 m.o. ** P = 0.0069; Sham vs. Vaccinated, ns P = 0.9995; Sham vs. 18 m.o. ** P = 0.0044; Vaccinated vs. 18 m.o. * P = 0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P = 0.1169; 7 m.o. vs. Vaccinated, ns P > 0.9999; 7 m.o. vs. 18 m.o. *** P = 0.0009; Sham vs. Vaccinated, ns P = 0.0577; Sham vs. 18 m.o. ** P = 0.007; Vaccinated vs. 18 m.o. **** P < 0.0001, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: The target genes NOTCH3 , Hes1 , Hey1 and Nrip2 on TgN3R182C150 mice at 5 and 12 months of age. Three biological replicates were run in three technical replicates. Error bars indicate standard deviation (SD).

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Standard Deviation

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF 1–5 ‐immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti‐perlecan antibody (green). Scale bar = 20 μm. 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‐month‐old TgN3R182C150 ( n = 3) and 7‐month‐old TgN3R182C150 ( n = 6) mice and 18‐month‐old TgN3R182C150 ( 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 = 8), and non‐vaccinated TgN3R182C150 ( n = 6) mice. Statistical significance was assessed using a Brown‐Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons (NOTCH3 ECD deposits (% of vessel area): 3 m.o. vs. 18 m.o. ** P = 0.0029; 3 m.o. vs. 7 m.o. *** P = 0.0004; 7 m.o. vs. Sham, ns P = 0.7326; 7 m.o. vs. Vaccinated, ** P = 0.003; Sham vs. Vaccinated, **** P < 0.0001. NOTCH3 ECD deposits (number/1,000 μm 2 ): 3 m.o. vs. 18 m.o. ** P = 0.0038; 3 m.o. vs. 7 m.o. *** P = 0.0002; 7 m.o. vs. Sham, ns P = 0.9913; 7 m.o. vs. Vaccinated, * P = 0.021; Sham vs. Vaccinated, *** P = 0.001. NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. * P = 0.0215; 3 m.o. vs. 7 m.o. ** P = 0.0029; 7 m.o. vs. Sham, ns P = 0.3622; 7 m.o. vs. Vaccinated, * P = 0.0428; Sham vs. Vaccinated, ** P = 0.0038, ns = non‐significant). Dotted lines indicate quartiles and dashed thicker lines are the median. Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: NOTCH3 ECD was detected in the whole blood serum of the non‐treated TgN3R182C150 mice at 3 months ( n = 9) of age and further increased at 7 months ( n = 6) of age. Serum from Notch3 −/− and C57Bl6/J WT mice were included as negative controls. Statistical analysis was performed using unpaired Student's t ‐test with Welch's correction (3 m.o. vs. 7 m.o. *** P = 0.0005). Error bars indicate standard error of the mean (SEM). NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated ( n = 10) TgN3R182C150 mice vs. sham ( n = 9). Statistical analysis was performed using unpaired Student's t ‐test with Welch's correction (Sham vs. Vaccinated * P = 0.0196). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques:

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Serum from 7‐month‐old non‐vaccinated TgN3R182C 150 mice ( n = 4) was monitored in the absence or presence of serum (1:1 dilution) from sham‐and vaccinated C57Bl6/J WT mice. No significant changes in NOTCH3 ECD concentration was observed after dilution adjustment. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Dunnett's multiple comparisons test (TgN3R182C 150 vs. TgN3R182C 150 + C57BL6/J WT Sham, ns P = 0.8383; TgN3R182C 150 vs. TgN3R182C 150 + C57BL6/J WT Vaccinated, ns P = 0.7000; ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Concentration Assay

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images show microglia stained with anti‐CD68 antibody (red) and Iba1 antibody (green). Scale bar = 20 μm. Quantification of CD68‐stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF 1–5 ‐immunized ( n = 6), sham ( n = 4), and non‐vaccinated TgN3R182C150 ( n = 5) mice. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Tukey's multiple comparisons test (% microglia with CD68 staining: 7 m.o. vs. Sham ns P = 0.7393; Sham vs. Vaccinated ** P = 0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P = 0.9131; Sham vs. Vaccinated * P = 0.0157, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Representative images of TgN3R182C150, sham‐, and NOTCH3 EGF 1–5 ‐immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar = 20 μm. Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF 1–5 ‐immunized ( n = 11), sham ( n = 8), and non‐vaccinated TgN3R182C150 ( n = 6) mice at 7 months of age. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Tukey's multiple comparisons test (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P = 0.7997; Sham vs. Vaccinated ns P = 0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P = 0.5362; Sham vs. Vaccinated ns P = 0.8059. NOTCH3 ECD deposits (number/1,000 μm 2 ): 7 m.o. vs. Sham ns P = 0.8392; Sham vs. Vaccinated ns P = 0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P = 0.6664; Sham vs. Vaccinated ns P = 0.8787, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: 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 TgN3R182C150 mice at 7 months of age. Scale bar = 50 μm. 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 TgN3R182C150 mice. Scale bar = 50 μm. Immunostaining for smooth muscle actin (ASMA) shows an extensive loss of VSMC in the Notch3 −/− mice when compared with a WT (C57Bl6/J) at 3 months of age. Scale bar = 50 μm.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Immunostaining

Journal: EMBO Molecular Medicine

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

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: NIH3T3 cells were transfected with the control, WT 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 (Control: Fc + DMSO vs. Jag2 + DMSO ns P = 0.7223; Jag2 + DMSO vs. Jag2 + DAPT ns P = 0.817. N3WT: Fc + DMSO vs. Jag2 + DMSO *** P = 0.0004; Jag2 + DMSO vs. Jag2 + DAPT ** P = 0.0069. N3R182C: Fc + DMSO vs. Jag2 + DMSO * P = 0.0135; Jag2 + DMSO vs. Jag2 + DAPT * P = 0.0352. Jag2 + DMSO: Control Jag2 + DMSO vs. N3WT Jag2 + DMSO *** P = 0.0008; Control Jag2 + DMSO vs. N3R182C Jag2 + DMSO * P = 0.0314; N3WT Jag2 + DMSO vs. N3R182C Jag2 + DMSO ns P = 0.4187, ns = non‐significant). Error bars indicate standard error of the mean (SEM). RLU, relative luminescence units. Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Transfection, Control, Cell Culture

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3 is overexpressed in multiple human tumors (A) Expression of NOTCH3 mRNA in primary human tumors from TCGA compared to normal tissues. Box and whiskers plots are drawn with individual points below 10 th and above 90 th percentiles. Median values are drawn as a line in the middle of the box. TPM, transcripts per kilobase million, ns, non-significant. (B) NOTCH3 mRNA fold change in primary human lung, breast, and ovarian tumors and xenografts compared to normal tissues (baseline, dashed line). Data represent mean (n = 2–4). (C) Quantitation of NOTCH3 ISH staining as a measure of percentage of area stained on breast (ER + , TNBC), lung (LUAD, LUSC), ovarian, and normal (breast, lung, and ovary) tissue sections.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Expressing, Quantitation Assay, Staining

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Generation and characterization of therapeutic anti-NOTCH3 mAbs (A) NOTCH3-dependent report gene assay with NOTCH3-targeted and control mAbs. Data represent mean ± SEM from 3 biological replicates (n = 4 per replicate), ns, non-significant. (B) qRT-PCR of HES1 gene expression in treated cells. Data represent mean ± SD from 2 biological replicates (n = 3 per replicate), ns, non-significant. (C) NOTCH3 immunoblot from mAb-treated cells and xenografts. GAPDH is shown as a loading control. Schematic diagram of NOTCH3 cleavage events and protein fragments. M, mouse number. (D and E) Immunoblot using N- or C-terminal domain antibodies to detect NOTCH3 fragments after DLL4 activation in treated cells. β-actin is shown as a loading control. (F) Epitope mapping of anti-NOTCH3 mAbs using NRR3-NRR1 domain swap chimeric constructs. NRR3 domains are shown in black and NRR1 domains are shown in gray. Representative data represent mean (n = 2). (G) Binding interface of anti-N3(i) Fab on the NOTCH3-NRR domain as determined from the co-crystal structure is shown in red, LNR-A (green), LNR-B (beige), LNR-C (blue), HD1 (pink), and HD2 (magenta).

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Gene Assay, Control, Quantitative RT-PCR, Gene Expression, Western Blot, Activation Assay, Construct, Binding Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Cell - membrane distribution of anti-NOTCH3 mAbs depends on signaling status (A) Confocal images of anti-NOTCH3 mAbs on fixed cells. Scale bar, 10 μm. (B) Single optical sections from live-cell confocal imaging of anti-NOTCH3 mAbs simultaneously bound to cells and imaged over time with anti-N3(i)-Alexa 488 (green) and anti-N3-DyLight647 (magenta). Scale bar, 10 μm. (C) z stack of maximum intensity projections from live-cell confocal imaging of anti-NOTCH3 mAbs over time. Scale bar, left = 8 μm, right = 10 μm. (D) Cross-section of cells bound by NOTCH3-targeted mAbs from images in Figure 2 C. Scale bar, 10 μm.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Membrane, Imaging

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs have differential rates of endocytosis and lysosomal trafficking (A and B) Colocalization of mAbs with pHrodo Red dextran compartments calculated by PCC and MCC from live-cell confocal images. (A) Data represent mean ± SEM of 3 biological replicates (n = 3 fields imaged per replicate). (B) Data represent mean ± SEM of 2 biological replicates (n = 2 fields imaged per replicate), ns, non-significant. (C) Colocalization of mAbs with CT-β were calculated by PCC and MCC from live-cell confocal images. Data represent mean ± SEM of 2 biological replicates (n = 5 fields imaged per replicate). (D) Single optical sections of mAbs with CT-β from live-cell confocal imaging. Scale bar, 10 μm. (E) Colocalization of mAbs with CAV-1 or clathrin were calculated by MCC in fixed cells. Data represent mean ± SEM (n = 3 fields imaged). (F) Colocalization of mAbs with pHrodo Red dextran compartments were calculated by MCC from live-cell confocal images of MβCD-treated cells. Cross hairs were set at a value of 0.2 MCC to highlight more rapid colocalization of both mAbs with pHrodo Red dextran compartments. Data represent mean ± SEM of 2 biological replicates (n = 5 fields imaged per replicate).

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Imaging

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Generated, Activity Assay, In Vitro, Control, Knockdown, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce prolonged tumor regressions (A) Tumor growth inhibition of HCC2429 CLXs treated with control and NOTCH3-targeted mAbs and ADCs. Data represent mean ± SEM (n = 7–8 per group). (B) Tumor growth inhibition of 37622 PDXs treated with NOTCH3-ADCs compared to cisplatin or control ADC. Data represent mean ± SEM (n = 9–10 per group). (C) Tumor growth inhibition of MDA-MB-468 CLXs treated with control and anti-N3 ADCs. Data represent mean ± SEM (n = 7–8 per group). (D) Tumor growth inhibition of OVCAR3 CLXs treated with control and anti-N3 ADCs compared with carboplatin alone, and carboplatin then anti-N3 ADC. Data represent mean ± SEM (n = 7–8 per group).

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Inhibition, Control

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Control, Polymer, Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Adjuvant, Antibody Labeling, RNAscope, Sequencing, Software

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: Histology of tumor invasive front. H&E staining. Scale bar, 100μm. B: Distribution of NOTCH3-positive fibroblastic cells. C: Distribution of α-SMA-positive fibroblastic cells. Cells stained brown in B and C represent positive cells for each antibody. Arrows in A , B and C indicate blood vessel layer, which is positive internal control of each antibody. D , E , F: Dual immunohistochemical analysis for α-SMA (red) and NOTCH3 (Green). Co-localization of α-SMA and NOTCH3 in fibroblasts was observed in cancer stroma. Scale bar, 50μm. Ca, cancer cells. Dotted lines in A - F indicate the interface of cancer nests and stroma. G: Kaplan-Meier curve for overall survival in relation to NOTCH3 expression in CAFs using 93 human OSCC cases. Log-rank test was used to calculate significance.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Control, Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: α-SMA and NOTCH3 expression in fibroblasts in the OSCC stroma.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: Clinico-pathological Significance of NOTCH3 expression in CAFs.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cultured alone (ctl; control) or cocultured with represented oral and maxillary SCC cell lines. 3 days after coculture, NHDFs were isolated from this coculture by using anti-Fibroblast microbeads for Western blot analysis. B and C: Immunofluorostainig using coculture with HO1-N-1 and NHDFs. Culture of NHDFs alone was used as a control. The bundles of NOTCH3-positive NHDFs (green) were observed around the AE1/AE3-positve HO1-N-1 cancer nests (red) ( B ). Double NOTCH3 and α-SMA-positive NHDFs were intervened between HO1-N-1 cancer nests. Ca, HO1-N-1 cancer nests. Dotted lines demonstrate the interface of HO1-N-1 cancer nests and NHDFs. ( C ). Scale bar, 100μm. The nuclei were counterstained by DAPI. D-G: NHDFs transfected with negative control siRNA (siCtrl) or siRNA for NOTCH3 (siN3) was cultured alone or cocultured with HO1-N-1 cells. 3 days after coculture, NHDFs were isolated from this coculture and subjected to western blot ( D ) and qPCR analyses to measure NOTCH3 ( E ), HEY1 ( F ), α-SMA ( G ). H: NHDFs were cultured alone (control), or directly cocultured with HO1-N-1 cells, or cocultured with transwell-separated HO1-N-1 cells. 3 days after culture, NHDFs were isolated and subjected to qPCR analysis. I: qPCR to measure each NOTCH mRNA expression in NHDFs isolated from coculture with HO1-N-1 cells. ND, not detected. * P < 0.05, ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Isolation, Western Blot, Transfection, Negative Control, Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: JAGGED1 expression in various oral cancer cell lines was analyzed by Western blot. B: The effect of NOTCH3 on cell proliferation. The proliferation of HO1-N-1 cells with or without recombinant human NOTCH3-Fc chimera treatment was monitored for 72hr.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A and B : Comparison of the microvessel density (MVD) between NOTCH3(-) CAFs and NOTCH3(+)CAFs cases. Immunofluorostaining for α-SMA (green), NOTCH3 (green) and CD34 (red) using human tongue OSCC samples. Ca, cancer nests. Dotted lines showed the interface of the cancer nests and stroma. Arrows demonstrated the CD34-positive microvessel. Scale bar, 50μm. The nuclei were counterstained by DAPI ( A ). Quantitative evaluation of MVD between these two groups ( B ). C-F : In vitro angiogenesis assay cocultured with HO1-N-1 cells, HUVECs and siRNA transfected NHDFs. Immunofluorostaining for NOTCH3 (green) and CD31 (red). Ca, HO1-N-1 cancer nests. Dotted lines indicate the margin of the HO1-N-1 cancer nests. Scale bar, 100μm. The nuclei were counterstained by DAPI ( C ). NHDFs isolated from this coculture were subjected to Western blot ( D ). Representative image of tube formation by HUVECs in each condition. Scale bar, 100μm ( E ). Quantitative evaluation of tube formation area (CD31-positive area) in each condition ( F ). siCtrl, negative control siRNA. siN3, siRNA for NOTCH3. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, In Vitro, Angiogenesis Assay, Transfection, Isolation, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cocultured with various cancer cell lines derived from non-oral lesions. 3 days after coculture, NHDFS were isolated from this coculture and subjected to Western blot analysis to detect NOTCH3 and α-SMA. B-D: In vitro angiogenesis assay cocultured with A549, HUVECs and siRNA transfected NHDFs. Representative images of tube formation ( B ) and its quantitative evaluation ( C ) in each condition. Scale bar, 100μm. NHDFs isolated from this angiogenesis assay were subjected to Western blot analysis ( D ). siCtrl, negative control siRNA. siN3, siRNA for NOTCH3. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Derivative Assay, Isolation, Western Blot, In Vitro, Angiogenesis Assay, Transfection, Negative Control

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cocultured with HO1-N-1 for 3 days. Prior to coculture, NHDFs were transfected with negative control siRNA or siRNA for NOTCH3. After 3 days, NHDFs were isolated from this coculture by using anti-fibroblast microbeads. NHDFs were subjected to real-time qPCR analyses to measure Notch3 expression. B-C: These isolated NHDFs were seeded on 24-well plates (2.5×10 5 cells/well) and cocultured with HUVECs (3.0×10 4 cells/well) for 4 days. Representative images of tube formation ( B ) and its quantitative evaluation ( C ) in each condition. Scale bar, 100μm. ctlNHDF(siCtl), negative control siRNA transfected NHDF isolated from culture alone. co-NHDF(siCtl), negative control siRNA transfected NHDF isolated from coculture with HO1-N-1. co-NHDF(siN3), siRNA for NOTCH3 transfected NHDF isolated from coculutre with HO1-N-1. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Negative Control, Isolation, Expressing