Journal: International Journal of Medical Sciences

Article Title: Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells

doi: 10.7150/ijms.43184

Figure Lengend Snippet: Sequences of primers used for RT-PCR.

Article Snippet: The following reagents and antibodies were used in the present study: anti-DLL4, anti-Hes-1, and anti-Hey-1 (Abcam, Cambridge, UK), anti-Jagged1, anti-Notch1, and anti-Notch3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β actin (Calbiochem, La Jolla, CA), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody and anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells

doi: 10.7150/ijms.43184

Figure Lengend Snippet: IS reduces Notch Signal activity in Dahl-hypertensive rats. Immunohistochemical analysis of Notch1 (a), Notch3 (b), and Hes-1 (c) in the aortas of DS, DH, and DH+IS rats (× 200 magnification, bar=50 µm), and calcification area of DH+IS rats (× 400 magnification, bar=50 µm).

Article Snippet: The following reagents and antibodies were used in the present study: anti-DLL4, anti-Hes-1, and anti-Hey-1 (Abcam, Cambridge, UK), anti-Jagged1, anti-Notch1, and anti-Notch3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β actin (Calbiochem, La Jolla, CA), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody and anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA).

Techniques: Activity Assay, Immunohistochemical staining

Journal: International Journal of Medical Sciences

Article Title: Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells

doi: 10.7150/ijms.43184

Figure Lengend Snippet: IS reduces the expression of Notch-related molecules in Dahl-hypertensive rats. Aortic mRNA and protein expression levels of Notch-related molecules in DS, DH, and DH+IS rats were analyzed by quantitative RT-PCR and western blotting; respectively. Values are mean ± SD (n=6-8). (a) Quantitative analysis of Jagged1 mRNA expression in aortic tissue. *p<0.0001 vs DS group, #p<0.0001 vs DH group. (b) Quantitative analysis of DLL4 mRNA expression in aortic tissue. *p<0.0007 vs DS group, #p<0.001 vs DH group. (c) Quantitative analysis of Notch1 mRNA expression in aortic tissue *p<0.005 vs DS group, #p<0.007 vs DH group. (d) Quantitative analysis of Notch3 mRNA expression in aortic tissue. *p<0.001 vs DS group, #p<0.003 vs DH group. (e) Quantitative analysis of Hes-1 mRNA expression in aortic tissue. *p<0.0001 vs DS group, #p<0.0001 vs DH group. (f) Quantitative analysis of Hey-1 mRNA expression in aortic tissue *p<0.004 vs DS group, #p<0.01 vs DH group. (g) Expression levels of representative proteins Jagged1, DLL4, Notch1, Notch3, Hes-1, and Hey-1 in the aorta of DS, DH, and DH+IS rats.

Article Snippet: The following reagents and antibodies were used in the present study: anti-DLL4, anti-Hes-1, and anti-Hey-1 (Abcam, Cambridge, UK), anti-Jagged1, anti-Notch1, and anti-Notch3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β actin (Calbiochem, La Jolla, CA), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody and anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells

doi: 10.7150/ijms.43184

Figure Lengend Snippet: IS reduces Notch1 and Notch3 expression in aortic smooth muscle cells (SMCs). Rat and human aortic SMCs were treated with IS for the indicated time intervals (a and e) and at the indicated doses (b and f). (a) IS increased the expression level of Notch1 mRNA after 24- and 48-hr incubation but the level after incubation for 96 hrs was lower than non-treated control (**p<0.002 vs non-treated control, #p<0.0001 vs non-treated control). (b) IS increased Notch1 mRNA expression when used at 250 and 500 μmol/L, but the level in the presence of 1000 μmol/L was lower than at 250 and 500 μmol/L (**p<0.0002 vs non-treated control, #p<0.001 vs 500 μmol/L group). (e and f) A similar trend was noted for the effect of duration of incubation with IS (*p<0.01 vs non-treated control, #p<0.01 vs non-treated control) and dose of IS (*p<0.01 vs non-treated control, #p<0.05 vs 500 μmol/L group) on Notch3 mRNA. Data are mean ± SD (n=4-6 per group). (c, d, g, and h) Effects of duration of incubation and dose of IS on Notch1 and Notch3 protein levels in aortic SMCs cell lysates measured by western immunoblotting using anti-Notch1 and anti-Notch3 antibodies, respectively. IS decreased (c and d) Notch1 and (g and h) Notch3 protein levels after 96h and at the concentration of 1000 μmol/L. Representative data of 5 similar experiments.

Article Snippet: The following reagents and antibodies were used in the present study: anti-DLL4, anti-Hes-1, and anti-Hey-1 (Abcam, Cambridge, UK), anti-Jagged1, anti-Notch1, and anti-Notch3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β actin (Calbiochem, La Jolla, CA), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody and anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA).

Techniques: Expressing, Incubation, Control, Western Blot, Concentration Assay

Journal: International Journal of Medical Sciences

Article Title: Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells

doi: 10.7150/ijms.43184

Figure Lengend Snippet: IS induces cellular calcium deposition in aortic SMCs. Calcium deposition in human aortic SMCs was induced by inorganic phosphate (3 mM). Cells were seeded at a density of 2×105 cells/well on 12-well culture plate in D-MEM containing 10% FBS for 48 hrs. Cells were pre-incubated with DAPT (20 μM) and ZVAD (100 μM) for 1 hr, then stimulated with Pi (3 mM) or IS (1000 μM) for 72 hrs. Absorbance was measured at 570~650 nm using a microplate reader. (a) Representative images showing calcium deposition in IS- or Pi-treated human aortic SMCs (×200 magnification, bar=50 µm). (b) Quantitative analysis of calcium deposition in IS- and Pi-treated human aortic SMCs. Data are mean±SD (n=6 per group). *p<0.009 vs Pi (3 mM) group, #p<0.0008 vs Pi (3 mM) group, †p<0.0059 vs DAPT-treated group, § p<0.009 vs IS-treated group. Expression levels of Notch-related and osteogenic-related mRNA with single knockdown of Notch1 and 3 or double knockdown of Notch1/Notch3 in rat aortic SMCs was analyzed by quantitative RT-PCR. Data are mean ± SD (n=6-8 per group). (c) Notch1 (**p<0.01 vs si-control group) (d) Notch3 (**p<0.01 vs si-control group) (e) Hes-1 (**p<0.01 vs si-control group) (f) Hey-1 (*p<0.05 vs si-control group, **p<0.01 vs si-control group) (g) Runx2 (*p<0.05 vs si-control group, **p<0.01 vs si-control group) (h) BMPS2 (*p<0.05 vs si-control group, **p<0.01 vs si-control group) (i) osteopontin (OPN) (*p<0.05 vs si-control group, **p<0.01 vs si-control group) (j) osteocalcin (OCN) (*p<0.05 vs si-control group, **p<0.01 vs si-control group), and (k) SM22 (*p<0.05 vs si-control group, **p<0.01 vs si-control group).

Article Snippet: The following reagents and antibodies were used in the present study: anti-DLL4, anti-Hes-1, and anti-Hey-1 (Abcam, Cambridge, UK), anti-Jagged1, anti-Notch1, and anti-Notch3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β actin (Calbiochem, La Jolla, CA), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody and anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA).

Techniques: Incubation, Expressing, Knockdown, Quantitative RT-PCR, 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 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: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: ZEB1 represses NOTCH3 , facilitating EMT and tumor initiation. a Serial transplantation experiments with DOX-inducible ICN3I- expressing TE11 (TE11- ICN3 TetOn ). Tumor formation rates were determined as in Fig. . * P < 0.05 vs. CD44L and DOX (−); n = 10. b Representative flow cytometry scatter plots of EPC2T cells with NOTCH3 -targeted or non-silencing (NS) control shRNA. NOTCH3 shRNA increased CD44H cells (lower right quadrant) to 29.8 ± 0.4% s.d. as compared to 0.4 ± 0.1% s.d. in NS control ( P < 0.0001 by Student’s t -test, n = 3). c qRT-PCR analysis for indicated genes comparing EPC2T cells with or without NOTCH3 knockdown. mRNA level for each gene in NS control cells was set to 1. * P < 0.05 vs. NS, n = 3. d Schematic of NOTCH3 second intron ( N3Int2 ) region and ChIP PCR primers in f and Supplementary Fig. . Primers amplify the region lacking ZEB1 or CSL-binding sites. e Transfection assays for pGL3-N3Int2-luc reporter activity with or without ectopic ZEB1 or ZEB2 expression. bla , empty vector control for ZEBs. * P < 0.05 vs. bla and pGL3-luc (empty reporter); # P < 0.05 vs. bla and pGL3-N3Int2-luc ; ns not significant vs. bla and pGL3-N3Int2-luc ; n = 4. f ChIP assays for ZEB1 binding to N3Int2 region in purified CD44L and CD44H cells. CD44L cells were stimulated with TGFβ for 14 days to induce CD44H cells. * P < 0.0005 vs. CD44L and IgG and TGFβ (−); # P < 0.005 vs. CD44H and IgG and TGFβ (−); ** P < 0.05 vs. CD44L and anti-ZEB1 and TGFβ (+); ns not significant vs. IgG and TGFβ (−); n = 3. g Representative flow cytometry scatter plots of EPC2T cells with ZEB 1-targeted or NS control shRNA. * P < 0.05 vs. NS and TGFβ (−); # P < 0.0001 NS and TGFβ (+); ns not significant vs. NS and TGFβ (−); n = 3. In b , g , flow cytometry was done 7 days following lentivirus infection. All bar diagrams represent mean ± s.d. At least three independent replicates were performed for all experiments. Fisher’s exact test was used for percentage comparisons in a . Student’s t -test was used for paired data comparisons in b , c , f . ANOVA with Tukey’s post hoc test was used for multiple comparisons in e , g

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Transplantation Assay, Expressing, Flow Cytometry, Control, shRNA, Quantitative RT-PCR, Knockdown, Binding Assay, Transfection, Activity Assay, Plasmid Preparation, Purification, Infection

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: NOTCH1 activation and ZEB1 expression in invasive ESCC predicts poor prognosis. a , b Representative IHC images for ICN1 and ZEB1 in ESCC cells in a deep invasive tumor nest and ESCC cells invading into lymphatic vessels. a ESCC #55; b ESCC #62 in Supplementary Data . c Representative IF image for ICN1 and CD44 in invasive ESCC cells. Scale bars, 50 μm in a – c . d Survival curve for 185 post-surgical ESCC patients with or without ICN1 expression at the tumor invasive front. e Model of the role of Notch1 in EMT in ESCC. Notch1 activation promotes tumorigenicity and heterogeneity in SCC via EMT. Notch1 drives squamous-cell differentiation by inducing Notch3 in normal squamous epithelia as well as SCC cells. Notch3 limits EMT. In response to TGFβ from the tumor microenvironment (e.g., cancer-associated fibroblasts and inflammatory cells), however, transcriptional repression of Notch3 by ZEB1 permits Notch1-mediated induction of CD44H cells via EMT. Notch activation may result in the generation and maintenance of CD44H cells possessing mesenchymal properties and enhanced malignant potential. EMT allows neoplastic cells to cope with stress during carcinogenesis and disease progression (e.g., genotoxic stress induced by 4NQO). CD44H cells produce pro-tumorigenic cytokines (e.g., IL-6) and tissue remodeling factors (e.g., MMP13, LOX, and POSTN) (Supplementary Fig. )

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Expressing, Cell Differentiation, Biomarker Discovery

Journal: Cell Death and Differentiation

Article Title: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

doi: 10.1038/cdd.2011.171

Figure Lengend Snippet: Upregulation of Notch signaling components in rhabdomyosarcoma (RMS) cell lines. ( a , left) Western blotting of Notch1-3 intracellular cleaved domains (IC), Jagged1, HES1 and β -actin (loading control) in whole-cell lysates from embryonal (ERMS) and alveolar (ARMS) RMS cell lines and normal human myoblasts SkMCs as control. Arrows indicate the IC domains of Notch1-3. Notch1 IC was detected using the antibody that recognizes only the activated form (cleaved at Val1744). ( a , right) Histograms report densitometric analysis of Notch1 IC (Val1744) , Notch2 IC , Notch3 IC , HES1, and Jagged1 bands normalized to β -actin of three independent experiments. ( b ) Western blot analysis of nuclear (N) and cytoplasmic (C) -enriched cell fractions of embryonal (ERMS) and alveolar (ARMS) RMS cell lines. Notch1 IC (Val1744) , Notch2 IC and Notch3 IC forms were detected in all cell lines. β -actin and topoisomerase II β were used as loading controls to discriminate the different cell fractions. ( c ) mRNA levels of Notch1-3, HES1 and Jagged1 (real time RT-PCR) were normalized to β -actin levels and expressed as fold increase over control SkMC (black column; 1 arbitrary unit). Columns, means; Bars, S.D. Results from three independent experiments are shown

Article Snippet: Short hairpin RNA (sh)RNA-mediated silencing was performed by transfecting cells with a pGFP-V-RS vector expressing 29 nucleotide shRNAs against Notch3 and a green fluorescent protein (GFP) as reporter gene (TG311139: Notch3 target sequence 5′-GTGAGAGCTGCAGAATATCGATGAC-3′ Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

doi: 10.1038/cdd.2011.171

Figure Lengend Snippet: Notch3 downregulation promotes RMS cell differentiation. ( a ) RD and RH30 cells cultured in complete medium (i.e., supplemented with 10% of fetal calf serum) were analyzed after 6 days of Notch3 or control (CTR) siRNA treatment. Representative immunofluorescence showing de novo expression of endogenous myosin heavy chain (MHC, green) in multinucleated fibers of Notch3 siRNA-transfected RD and RH30 cells (white arrows). Representative of three assays. ( b ) Western blotting showing de novo expression of Troponin I and β -actin (loading control) in Notch3 siRNA RD and RH30 cells treated as in ( a ). ( c ) Representative light microscopy pictures of RD and RH30 cells showing elongated multinucleated structures in Notch3 siRNA-treated cells treated as in ( a ). ( d ) Western blotting showing levels of Notch3 IC and HES1 (left) and Myogenin along with the phosphorylation of p38, Akt and mTOR (right) in RD and RH30 cells 24 and 48 h after CTR or Notch3 siRNA transfection. β -actin was the loading control. Representative of three independent experiments. ( e ) Western blotting showing levels of Notch3 IC and HES1 in RD and RH30 cells 24 and 48 h after CTR or Jagged1 siRNA transfection. β -actin was the loading control. Representative of three independent experiments

Article Snippet: Short hairpin RNA (sh)RNA-mediated silencing was performed by transfecting cells with a pGFP-V-RS vector expressing 29 nucleotide shRNAs against Notch3 and a green fluorescent protein (GFP) as reporter gene (TG311139: Notch3 target sequence 5′-GTGAGAGCTGCAGAATATCGATGAC-3′ Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen).

Techniques: Cell Differentiation, Cell Culture, Immunofluorescence, Expressing, Transfection, Western Blot, Light Microscopy

Journal: Cell Death and Differentiation

Article Title: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

doi: 10.1038/cdd.2011.171

Figure Lengend Snippet: Notch3 depletion reduces proliferation and impairs RMS cell lines tumourigenic features in vitro . ( a ) RD and RH30 cells were transfected (t0) with Notch3 siRNA or control (CTR) siRNA, cultured in complete medium (i.e. supplemented with 10% of fetal calf serum) and harvested and counted at the indicated time points. * P <0.05; Bars , SD. ( b ) 2 × 10 4 RD living cells (evidenced by trypan blue exclusion) were seeded on soft-agar in 35 mm petri dishes 48 h after transfection with CTR or Notch3 siRNA. Histogram represents the mean number of colonies per field counted under a light inverted microscope 4 weeks after seeding (two independent experiments in triplicate). At least five fields per dish were checked at 100 × magnification. Bars , S.D. ** P <0.001

Article Snippet: Short hairpin RNA (sh)RNA-mediated silencing was performed by transfecting cells with a pGFP-V-RS vector expressing 29 nucleotide shRNAs against Notch3 and a green fluorescent protein (GFP) as reporter gene (TG311139: Notch3 target sequence 5′-GTGAGAGCTGCAGAATATCGATGAC-3′ Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen).

Techniques: In Vitro, Transfection, Cell Culture, Inverted Microscopy

Journal: Cell Death and Differentiation

Article Title: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

doi: 10.1038/cdd.2011.171

Figure Lengend Snippet: Notch3 downregulation prevents the G1-to-S phase transition in RMS cell lines. ( a ) RD and RH30 cells were transfected with Notch3 siRNA or control (CTR) siRNA and, 48 h later, stained with 5-ethynyl-2′-deoxyuridine (EdU)-Alexa 488/Cell cycle 633 for analysis by flow cytometry gating on living cells. Left, representative diagrams. Right, the histogram depicts the percentage of control (CTR) or Notch3 siRNA transfected RD and RH30 cells in G1, S and G2 phases. Representative of three independent experiments in duplicate. ( b and c ) Western blotting showing expression of p21 Cip1 , PTEN and the Ser 380 phophorylated form of PTEN, and pRb along with the phosphorylation of ERK1/2 in whole-cell extracts after CTR or Notch3 siRNA transfection. Arrows ( c ) point to bands corresponding to hyper- (upper) and hypo-phosphorylated (lower) pRb species. β -actin was the loading control

Article Snippet: Short hairpin RNA (sh)RNA-mediated silencing was performed by transfecting cells with a pGFP-V-RS vector expressing 29 nucleotide shRNAs against Notch3 and a green fluorescent protein (GFP) as reporter gene (TG311139: Notch3 target sequence 5′-GTGAGAGCTGCAGAATATCGATGAC-3′ Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen).

Techniques: Sublimation, Transfection, Staining, Flow Cytometry, Western Blot, Expressing

Journal: Cell Death and Differentiation

Article Title: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

doi: 10.1038/cdd.2011.171

Figure Lengend Snippet: HES1 downregulation in RMS cells induces cell differentiation mimicking the effect of Notch3 knockdown. RD and RH30 cells were transfected with a Notch3 or control (CTR) siRNA, and 24 h later they were transfected with a HES1 or control (CTR) siRNA * in complete medium (i.e., supplemented with 10% of fetal calf serum). ( a ) Western blotting showing expression of p21 Cip1 and Myogenin 48 h after HES1 silencing in whole-cell extracts. ( b ) Immunofluorescence analysis shows de novo expression of endogenous myosin heavy chain (MHC, red) in multinucleated fibers of Notch3, HES1 and Notch3 plus HES1 siRNA-transfected RD and RH30 cells after 6 days. Representative images of three assays

Article Snippet: Short hairpin RNA (sh)RNA-mediated silencing was performed by transfecting cells with a pGFP-V-RS vector expressing 29 nucleotide shRNAs against Notch3 and a green fluorescent protein (GFP) as reporter gene (TG311139: Notch3 target sequence 5′-GTGAGAGCTGCAGAATATCGATGAC-3′ Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen).

Techniques: Cell Differentiation, Transfection, Western Blot, Expressing, Immunofluorescence

Journal: Cell Death and Differentiation

Article Title: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

doi: 10.1038/cdd.2011.171

Figure Lengend Snippet: HES1 overexpression in RMS cells abrogates the effects of Notch3 knockdown. RD and RH30 cells were infected with an Adenovirus expressing HES1 (AdHES1) or a control Adenovirus (Ad-GFP) 24 h after transfection with CTR or Notch3 siRNAs. ( a ) Western blotting showing levels of Notch3 IC , HES1, Myogenin and p21 Cip1 24 h and 48 h after Adenovirus infection. β -actin and α -tubulin were the loading controls. Representative of three independent experiments. ( b ) Cell cycle analysis by propidium iodide of RD and RH30 cells 48 h after adenovirus infection with AdHES1 and AdGFP. Left, representative diagrams. Right, histograms depict the fold change of the cell percentage in the different cell cycle phases for AdHES1 versus AdGFP infected RD and RH30 cells, after normalization for the efficiency of infection . ( c ) Immunofluorescence analysis of AdHES1- and AdGFP-infected RD and RH30 cells (GFP, green) with anti-myosin heavy chain (MHC, red) antibody 6 days after infection. Representative images of three independent experiments

Article Snippet: Short hairpin RNA (sh)RNA-mediated silencing was performed by transfecting cells with a pGFP-V-RS vector expressing 29 nucleotide shRNAs against Notch3 and a green fluorescent protein (GFP) as reporter gene (TG311139: Notch3 target sequence 5′-GTGAGAGCTGCAGAATATCGATGAC-3′ Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen).

Techniques: Over Expression, Infection, Expressing, Transfection, Western Blot, Cell Cycle Assay, Immunofluorescence

Journal: Cell Death and Differentiation

Article Title: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

doi: 10.1038/cdd.2011.171

Figure Lengend Snippet: Notch3 downregulation in RMS cells reduces tumour growth in vivo . ( A ) RH-30 cells were transfected with a Notch3 short hairpin (sh)RNA- or control (CTR) shRNA-GFP-plasmids and images of GFP fluorescence (grey) were acquired after 3 weeks of puromycin selection (Left). Arrows depict nuclei of newly formed myofibers (200 × magnification). (Right) 48 h post-shRNAs transfection, positive GFP was separated from negative GFP cells by cell sorting and expression of Notch3 IC , and β -actin (loading control) were analyzed by western blotting. Representative of two independent experiments. ( B , Left) Mouse-bearing CTR shRNA (as a mixture of control (CTR) shRNA-GFP/wild-type cells) and Notch3 shRNA (as a mixture of Notch3 shRNA-GFP/wild-type cells) tumour xenografts (black arrows; left flank : a and right flank: f , respectively). (Middle) xenografts from nude mice injected with Notch3 shRNA RH30 cells (wild-type/Notch3 shRNA cell ratio ∼60%/40% f, g, h, i and l ). Xenografts from CTR shRNA cells (wild-type/CTR shRNA cell ratio ∼50%/50% a, b, c, d and e ) were controls. (Right) Histogram reports tumour volumes of each xenograft. ( C , Left) Haematoxylin/eosin staining and immunolabeling of Ki67 and GFP of 5 μ m serial sections from xenografts of mice injected with CTR shRNA and Notch3 shRNA RH30 cells (from d and i samples) (400 × magnification). GFP right panels are a higher magnification of left panels (600 × magnification). Representative of three xenografts per condition. ( D ) Histogram depicts the average of the percentage of GFP-positive cells per field in five fields per tumour section. Bars , S.D. * P <0.05. ( E ) The expression of Notch3 IC was analysed by western blotting in lysates of samples from mice injected with CTR shRNA and Notch3 shRNA RH30 cell suspensions (three xenografts were pooled per group)

Article Snippet: Short hairpin RNA (sh)RNA-mediated silencing was performed by transfecting cells with a pGFP-V-RS vector expressing 29 nucleotide shRNAs against Notch3 and a green fluorescent protein (GFP) as reporter gene (TG311139: Notch3 target sequence 5′-GTGAGAGCTGCAGAATATCGATGAC-3′ Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen).

Techniques: In Vivo, Transfection, shRNA, Fluorescence, Selection, FACS, Expressing, Western Blot, Injection, Staining, Immunolabeling