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: For Notch3 extracellular staining, cells were incubated with murine Notch3 antibody (R&D Systems; Cat#AF1308) or normal goat IgG (R&D Systems; Cat#AB-108-C) used as a negative 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: For Notch3 extracellular staining, cells were incubated with murine Notch3 antibody (R&D Systems; Cat#AF1308) or normal goat IgG (R&D Systems; Cat#AB-108-C) used as a negative 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: For Notch3 extracellular staining, cells were incubated with murine Notch3 antibody (R&D Systems; Cat#AF1308) or normal goat IgG (R&D Systems; Cat#AB-108-C) used as a negative 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: For Notch3 extracellular staining, cells were incubated with murine Notch3 antibody (R&D Systems; Cat#AF1308) or normal goat IgG (R&D Systems; Cat#AB-108-C) used as a negative 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: For Notch3 extracellular staining, cells were incubated with murine Notch3 antibody (R&D Systems; Cat#AF1308) or normal goat IgG (R&D Systems; Cat#AB-108-C) used as a negative 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: For Notch3 extracellular staining, cells were incubated with murine Notch3 antibody (R&D Systems; Cat#AF1308) or normal goat IgG (R&D Systems; Cat#AB-108-C) used as a negative control.

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

Journal: Methods (San Diego, Calif.)

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

doi: 10.1016/j.ymeth.2012.07.008

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

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

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

Journal: Methods (San Diego, Calif.)

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

doi: 10.1016/j.ymeth.2012.07.008

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

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

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

Journal: bioRxiv

Article Title: NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy

doi: 10.1101/2023.07.10.548456

Figure Lengend Snippet: a , Deconvolution of NOTCH3+ meningioma mural cells from using human meningiomas with paired RNA sequencing and DNA methylation profiling (n=502). ANOVA. b , Immunoblots showing NOTCH3 is expressed in CH-157MN and IOMM-Lee Immune-enriched meningioma cell lines. c , In vivo tumor initiating capacity of CH-157MN meningioma cells in NU/NU mice ± αNRR3 IP injection 2 times per week. Denominators indicate number of mice at each time point. Numerators indicate number of mice with tumors at each time point. d , QPCR for the NOTCH3 target gene HEY1 from meningioma xenografts ± αNRR3 treatment for 2 weeks. Student’s t test. e , IHC for Ki-67 in meningioma xenografts showing αNRR3 blocks meningioma cell proliferation. Representative of n=3 xenografts per condition. Scale bar, 100μm. f , CH-157MN meningioma xenograft growth (left, student’s t tests) or survival (log-rank test). Arrows indicate initiation of bi-weekly treatment with the indicated therapy, which continued until death. g , IOMM-Lee meningioma xenograft growth (left, student’s t tests) or survival (log-rank test). Arrows as in f . h , QPCR for NOTCH3 or HEY1 in CH-157MN meningioma cells ± stable expression of empty vector (EV) or NOTCH3 ICD . Student’s t tests. i , IF quantification of the stem cell marker PTPRZ1 in CH-157MN meningioma cells. Student’s t test. j , Clonogenic in vitro growth of CH-157MN meningioma cells after 2 weeks. Student’s t test. k , In vivo tumor initiating capacity of CH-157MN meningioma cells ± EV or NOTCH3 ICD over limiting dilutions. Numerator and denominator as in c . l , CH-157MN meningioma xenograft growth (left, student’s t tests) or survival (log-rank test). m , Images of heterotopic meningioma xenografts showing macroscopic necrosis and ulceration in EV meningiomas. Representative of n=7–9 xenografts per condition. n , H&E low and high (box) magnification images of meningioma xenografts showing microscopic necrosis in EV meningiomas. Representative of n=3 xenografts per condition. Scale bars, 100μm. o , IHC for endothelia markers in meningioma xenografts showing NOTCH3 ICD induces meningioma angiogenesis. Representative of n=3 xenografts per condition. Scale bars, 100μm. Lines represent means and error bars represent standard error of means. **p≤0.01, ***p≤0.0001.

Article Snippet: NOTCH3 negative regulatory region neutralizing antibody treatments (αNRR3) and NOTCH1 negative regulatory region neutralizing antibody (αNRR1) treatments using murine antibodies from Genentech were performed as previously described , , with bi-weekly IP injection of 20mg/kg α NRR3, 10mg/kg α NRR1 (dose-reduced due to gastrointestinal and cutaneous toxicity leading to weight loss), or 20mg/kg IgG2a isotype control (BE0085, Bio X-cell). γ-secretase inhibitor treatments were performed using LY-411575 (SML0649, Millipore Sigma) as previously described , with daily 20μM/kg IP injections in 0.5% methylcellulose and 0.1% Tween 80 in 1xPBS.

Techniques: RNA Sequencing, DNA Methylation Assay, Western Blot, In Vivo, Injection, Expressing, Plasmid Preparation, Marker, In Vitro

Journal: bioRxiv

Article Title: NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy

doi: 10.1101/2023.07.10.548456

Figure Lengend Snippet: a , Network of gene circuits distinguishing recurrent (n=99) from primary (n=403) human meningiomas using RNA sequencing. Nodes represent pathways and edges represent shared genes between pathways (p≤0.01, FDR≤0.01). Red nodes are enriched and blue nodes are suppressed in recurrent versus primary meningiomas. b , IHC for Ki-67 in recurrent (n=53) versus primary (n=123) meningiomas, or RNA sequencing of recurrent (n=99) versus primary (n=403) meningiomas for deconvolution of NOTCH3+ meningioma mural cells from or quantification of NOTCH3 or HEY1 expression. TPM, transcripts per million. ANOVA. c , Multiplexed seqIF microscopy showing human meningioma recurrence after radiotherapy (RT) is associated with increased NOTCH3 and Ki-67. Many NOTCH3+ cells also express the interferon and innate immune regulators STING and pSTAT3. CD31 marks pericytes, COL1A marks fibroblasts, SSTR2A marks meningioma cells, and DAPI marks DNA. Representative of n=4 pairs of patient-matched primary and recurrent meningiomas. Scale bar, 100μm. d , CH-157MN meningioma xenograft growth (left and middle, student’s t tests) or survival (log-rank test) after expression of empty vector (EV) or NOTCH3 ICD ± RT showing NOTCH3 drives resistance to RT. Arrows indicate RT treatments (2Gy × 5 daily fractions). Xenografts from all arms were isolated for single-cell RNA-sequencing 1 day after completing RT (early) or once median survival was reached in the EV + RT arm (late). e , Single-cell RNA sequencing UMAP of 152,464 meningioma xenograft human cell transcriptomes showing tumor cell states ± αNRR3 treatment for 2 weeks as in or ± NOTCH3 ICD ± RT as in d . f , UMAP showing single-cell RNA sequencing of meningioma xenograft human cells shaded by experimental condition or phase of the cell cycle. g , Analysis of C2 G2M/S phase meningioma xenograft human cells in control versus NOTCH3 ICD versus αNRR3 conditions showing NOTCH3 drives meningioma cell proliferation. Colors as in f . Student’s t tests. h , Cell cycle analysis across all clusters of meningioma xenograft human cells ± NOTCH3 ICD ± RT showing NOTCH3 sustains cell proliferation through G2M and S phase despite RT. Student’s t test. i , Meningioma xenograft growth (left, student’s t tests) or survival (log-rank test) after treatment with RT as in d ± αNRR3 as in . αNRR3 treatment was initiated on the first day of radiotherapy and continued until death. Lines represent means and error bars represent standard error of means. *p<0.05, **p≤0.01.

Article Snippet: NOTCH3 negative regulatory region neutralizing antibody treatments (αNRR3) and NOTCH1 negative regulatory region neutralizing antibody (αNRR1) treatments using murine antibodies from Genentech were performed as previously described , , with bi-weekly IP injection of 20mg/kg α NRR3, 10mg/kg α NRR1 (dose-reduced due to gastrointestinal and cutaneous toxicity leading to weight loss), or 20mg/kg IgG2a isotype control (BE0085, Bio X-cell). γ-secretase inhibitor treatments were performed using LY-411575 (SML0649, Millipore Sigma) as previously described , with daily 20μM/kg IP injections in 0.5% methylcellulose and 0.1% Tween 80 in 1xPBS.

Techniques: RNA Sequencing, Expressing, Microscopy, Plasmid Preparation, Isolation, Control, Cell Cycle Assay