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: The Journal of Cell Biology

Article Title: Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains

doi: 10.1083/jcb.202211041

Figure Lengend Snippet: C-terminal Notch activating mutations signal by alternative mechanisms. (A) Schematic diagram showing the intracellular domain of Drosophila WT Notch and mutant constructs used, PPxF, ΔPEST, and ANK. NB all constructs have intact ECD (not shown). (B) Overactivation of the mutant Notch constructs and WT+Dx, analyzed by NRE-luciferase assay in S2 cells. ***P < 0.001 compared to WT Notch. (C) Activation of WT Notch and mutant constructs with and without treatment of cells with BB-94, MβCD, or ML-SI1. *, **, and *** indicate P < 0.05, 0.01, and 0.001 by two-tailed t test, respectively for comparisons indicated on the graph. Error bars represent SEM. Sample sizes are indicated in figure. (D) Time course of colocalization between Notch ECD antibody uptake and Cav-1-mRFP. Notch ECD antibody was labeled for 15 min (pulse), then chased for 0, 10, 30, and 60 min, and the percentage of Notch ECD on Cav1-mRFP positive spots was scored with WT Notch, PPxF, or ANK expressing cells. Data from 3 experimental repeats, with 50–100 puncta scored per repeat, error bars SEM.

Article Snippet: Primary antibodies used in this study were sheep anti-NOTCH3 ECD (1:500; R&D systems), rabbit anti-NOTCH3 ICD (D11B8, 1:500; Cell Signaling Technology), mouse anti-EEA1 (E9Q6G, 1:200; Cell Signaling Technology), and rabbit anti-clathrin heavy chain (Cat# 4796, RRID:AB_10828486; Cell Signaling Technology, used 1:50).

Techniques: Mutagenesis, Construct, Luciferase, Activation Assay, Two Tailed Test, Labeling, Expressing

Journal: Nature

Article Title: Human blood vessel organoids as a model of diabetic vasculopathy

doi: 10.1038/s41586-018-0858-8

Figure Lengend Snippet: a, Representative images of Col IV in diabetic blood vessel organoids (NC8), treated with antibodies against Jagged-1, Notch1, Notch3, or recombinant Dll1 and Dll4. The thickness of the Col IV+ coat of vessels was measured in optical cross-sections. Non-diabetic n=132, Vehicle n=139, α-Jagged1 n=141, Dll1 n=132, Dll4 n=153, α-Notch1 n=156, α-Notch3 n=156 lumina were analysed from 3 independent biological replicates with equal sample size. Mean ± SD. *** p<0.0001 (One-way ANOVA). b, Dermal blood vessels of T2D patients (diabetic) and healthy controls (non-diabetic) were stained for Notch3 and SMA. c, Hes5 and SMA were co-stained in dermal sections of T2D patients (diabetic) and non-diabetic controls. Note the strong Hes5 signal in pericytes (SMA) of diabetic patients. d,e, Quantification of Notch3 and Hes5 expression data from c and d. Mean ± S.E.M of n = (non-diabetic =4, diabetic=4) independent patients. * p=0.043 two-tailed student t-test. Scale bars a=50μm, b,c=20μm.

Article Snippet: Anti-Notch3 blocking antibodies (R&D AF1559) were injected 3 time/week at 1mg/kg.

Techniques: Recombinant, Staining, Expressing, Two Tailed Test

Journal: Nature

Article Title: Human blood vessel organoids as a model of diabetic vasculopathy

doi: 10.1038/s41586-018-0858-8

Figure Lengend Snippet: a, Representative images of Col IV in diabetic blood vessel organoids (NC8), treated with antibodies against Jagged-1, Notch1, Notch3, or recombinant Dll1 and Dll4. The thickness of the Col IV+ coat of vessels was measured in optical cross-sections. Non-diabetic n=132, Vehicle n=139, α-Jagged1 n=141, Dll1 n=132, Dll4 n=153, α-Notch1 n=156, α-Notch3 n=156 lumina were analysed from 3 independent biological replicates with equal sample size. Mean ± SD. *** p<0.0001 (One-way ANOVA). b, Dermal blood vessels of T2D patients (diabetic) and healthy controls (non-diabetic) were stained for Notch3 and SMA. c, Hes5 and SMA were co-stained in dermal sections of T2D patients (diabetic) and non-diabetic controls. Note the strong Hes5 signal in pericytes (SMA) of diabetic patients. d,e, Quantification of Notch3 and Hes5 expression data from c and d. Mean ± S.E.M of n = (non-diabetic =4, diabetic=4) independent patients. * p=0.043 two-tailed student t-test. Scale bars a=50μm, b,c=20μm.

Article Snippet: For blocking Notch receptors/ligands anti-Notch1 (Biolegend 352104, 10μg/mL), anti-Notch3 (R&D AF1559, 5μg/mL), anti-Jagged1 (R&D MAB12771, 5μg/mL) blocking antibodies were used.

Techniques: Recombinant, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Modular structure of NOTCH3, SP indicates signal peptide, red shaded EGF-like modules indicate core ligand binding region, LNR indicates Lin12-Notch repeats, NRR is the negative regulatory region, S1, S2 and S3 are proteolytic cleavage sites, HD-N and HD-C are the heterodimer interface region. RAM and Ankyrin repeat region (Ank) are motifs involved in binding CSL transcription factors, PEST domain is involved in ubiquitin-dependent turnover of the ICD. Location of mutants used in this study are indicated by arrows. Coloured bars indicate locations of regions of NOTCH3 in which epitopes used in this study are located. (B-D) AlphaFold2 predictions of WT NOTCH3 EGF modules 2 (B) , 5 (C) and 11 (D) , and mutant substitutions introduced using the SDM programme. Location of the new Cys residue in R90C indicated in yellow, and other substitutions that replace as cysteine residues are shown in magenta. (E-G) Surface views of the structures from (B-D) depicted as a surface mesh. The introduced cysteine in R90C is surface exposed (E) , but the free cysteines in C212Y and C455R are buried within the module structure. ΔΔG values indicate predicted decreases in stability. (H-K) Expressed WT NOTCH3 (H) and CADASIL mutants (I-K) do not show strong accumulation in the ER when expressed in hTert-RPE1 cells. (L, M) scoring of NOTCH3 localisation in KDEL-positive ER, by % area of ER occupied (L) and by % of total NOTCH3 staining intensity (K) in z-sections through the cytoplasmic region. R90C shows a small increase in %NOTCH3 which is localised to the ER. * indicates P<0.05 by student t-test, error bars SEM, n=10. (N) Antibody surface labelling of NOTCH3 localisation on non-permeabilised cells at time 0 and 60 minutes during a live cell anti-ECD uptake assay. Similar time zero surface distributions, and subsequent surface depletion of WT and CADASIL mutants can be seen.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Ligand Binding Assay, Binding Assay, Mutagenesis, Residue, Staining

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A-C) Schematic view of pulse chase labelling protocol. (D-G) WT NOTCH3 localisation in permeabilised cells after antibody uptake at 0 (D) , 15 (E) , 30 (F) , and 60 (G) minutes. Little surface-labelled NOTCH3 is transported to the early endosome by 15 minutes chase but is evident in EEA1 positive endosomes by 30 and 60 minutes. Presence of both full-length and ECD-only staining suggests ECD-shedding can occur by the time NOTCH3 is localised to the EEA1-marked endosome. (H-J) CADASIL mutant NOTCH3 localisation after 60-minute chase shows the mutant proteins labelled at the cell surface also become localised to the EEA1-positive endosome. (K, L) After 60-minute chase surface, little labelled NOTCH3 ECD has reached the CD63-positive late endosome while ICD is present. However full-length NOTCH3 and ECD-only localisations are found adjacent to CD63-marked organelles.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Pulse Chase, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A-D) Immunolocalisation of NOTCH3 with anti-ECD and anti-ICD compared with EEA1-marked endosomes in fixed and permeabilised cells of WT (A) and CADASIL mutants ( B-D) . (E) Quantitation of NOTCH3 localisation in the early endosome, showing % endosomal-located NOTCH3 puncta which either full-length NOTCH3, ECD-only, or ICD-only staining. R90C is distinguished from WT and other mutants by a greater proportion of ICD-only puncta compared to ECD or full-length. For WT, n=91 NOTCH3 puncta from198 endosomes scored. For R90C, n=139 NOTCH3 puncta from 180 endosomes score. For C212Y, n=140 NOTCH3 puncta from 240 endosomes scored. For C455R, n=164 NOTCH3 puncta from 302 endosomes scored. Minimum of 7 cells scored for each construct. Data obtained from z sections from at least 7 cells. (F) Full-length NOTCH3, or ECD-only puncta located within or adjacent to Rab11-positive endosomes. (G, H) In CD63 positive endosomes (G) and LAMP1-positive lysosomes (H) , NOTCH3 localisation is predominantly as ICD-only puncta, quantified in (I, J) . Error bars in I and J are SEM, n=11 and n=7 respectively. Statistical significance by student t-test.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Quantitation Assay, Staining, Construct

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Schematic diagram illustrating alternative explanations for visualisation of separated ECD and ICD-positive puncta, including: ECD shedding by S2 cleavage; epitope masking; fragmentation to remove terminal epitopes; non-specific staining. (B) Colocalisation of two different ECD epitopes compared to ICD ab23426 . ICD-stained puncta can be observed which lack both ECD epitopes. (C) Colocalisation of two different ICD epitopes compared to anti-ECD 1E . ECD-only spots are present which lack both ICD epitopes. (D, E) Quantification of the colocalisation between the epitopes used in B, C. In D * indicates P<0.05 compared with double ECD epitope colocalisation by student t-test. Error bars represent SEM, n=5 (D) , n=14 (E) . (F-H) Both ICD epitopes are located to the nucleus in NOTCH3 transfected cells but not the ECD epitope.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) EEA1-positive early endosomes of MCF7 cells costained with anti-ECD, and ICD. Arrowheads indicate endosomes shown enlarged in insets which have separate localisation of ECD and ICD puncta. (B) CD63-positive late endosomes costained with anti-ECD, and ICD. Boxed region is enlarged in inset showing late endosomes predominantly contain ICD-only puncta. (C) After treatment of MCF7 cells with metalloprotease inhibitor BB94, more full-length NOTCH3 staining is observed in the late endosomes, colocalised with CD63. (D) Treatment of cells with gamma-secretase inhibitor DAPT does not alter localisation of ECD in the late endosome. (E-G) Quantification of colocalization of ECD vs ICD (E) , ECD vs CD63 (F) , and ICD vs CD63 ( G) . Error bars, SEM, control DMSO-only treated cells n=10, BB94 n=18, DAPT n=12. * indicates p<0.05 by student t-test compared to control.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Schematic figure illustrating luciferase reporter assay methodology. (B) WT and CADASIL mutant NOTCH3 signalling after transfection into hTERT-RPE1 cells compared to empty vector (EV) control. * indicates p<0.05 compared to WT NOTCH3 by student t-test. Error bars SEM, minimum of n=4. (C-F) NOTCH3 signalling after treatment of transfected cells with TRPML inhibitor (ML-Sl1), gamma-secretase inhibitor (RO 4929097 ), or metalloprotease inhibitor (BB94) for WT (C) , R90C (D) , C212Y (E) , and C455R (F) * indicates p<0.05 compared with control by student t-test. Error bars are SEM, minimum of n=3.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Luciferase, Reporter Assay, Mutagenesis, Transfection, Plasmid Preparation, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Elabela gene therapy promotes angiogenesis after myocardial infarction

doi: 10.1111/jcmm.16814

Figure Lengend Snippet: ELA gene therapy increases VEGF/VEFGR2 and Jagged1/Notch3 expression in mice with myocardial infarction. AAV was injected into the surrounding myocardium and tail vein immediately after the model was established. Then, AAV was injected again from the tail vein one week later. The expression of apelin peptide jejunum (APJ) receptor (A and B), vascular endothelial growth factor (VEGF, A and C), VEGFR2 (A and D), Jagged1 (A and E) and Notch3 (A and F) from the indicated group (each group, n = 5) was detected by Western blot assay at 4 weeks after operation. Values are mean ± SEM. * p < 0.05, compared with the sham group;** p < 0.01, compared with the MI+PBS group

Article Snippet: After blocking with 5% skim milk, the membrane was incubated with TBS‐T–diluted primary antibody [rabbit anti‐mouse Flag, VEGF, VEGFR2, Jagged1 (Cell Signaling Technology), Notch3 (ProteinTech Group) and GAPDH (Abcam)] at 4°C overnight.

Techniques: Expressing, Injection, Western Blot