Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Time- and dose-dependent inhibition of NAC on the intracellular domain of Notch3 (N3IC), but not Notch1 (N1IC). HeLa cells were treated with NAC (2-10 mM) for 0-24 h. B. Dose-dependent inhibition by NAC (0-10 mM, 6 h) on the protein expression of N3IC in HeLa cells. C. NAC treatment (5 mM, 0-12 h) reduces protein levels of N3IC and extracellular domain of Notch 3 (N3EC) but not full length Notch 3 precursor (N3FL) in HeLa cells. Densitometry quantifications of the protein bands were shown after normalization with their respective β-actin levels. Data are presented as means ± SE, n=3. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (2-10 mM, 0-24 h) decreases Hes1 and HRT1 protein levels in HeLa cells. B. NAC treatment (0-10 mM for 6 h or 5 mM for 0-12 h) decreases Hes1 and HRT1 mRNA expression in HeLa cells. The mRNA expression of NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. C. NAC treatment (0-10 mM, 12 h) inhibits Hes1 reporter activity in HeLa cells. The luciferase activity in NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. D. Notch3 siRNA knockdown reduces Hes1 and HRT1 levels in HeLa cells. Protein levels were determined 2 days after siRNA transfection. siCtrl, scramble siRNA; siNotch3, Notch3 siRNA. Protein densitometry quantifications were shown after normalization with β-actin levels. Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Activity Assay, Luciferase, Knockdown, Transfection

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Pre-treatment with a γ-secretase inhibitor, DAPT (20 μM, 30 min), had no effect on NAC-induced (5 mM, 0-12 h) decrease in N3IC protein expression in HeLa cells. B. NAC treatment (5 mM, 0-12 h) did not affect Notch3 mRNA expression in HeLa cells. C. Pre-treatment with NH 4 Cl (25 mM, 1 h), but not lactacystin (10 μM, 30 min), reversed NAC-induced (5 mM, 0-12 h) decrease of N3IC protein levels in HeLa cells. D. NAC treatment did not affect levels of exogenously expressed Notch3 active intracellular domain (N3ICD). HeLa cells were transfected with vectors expressing N3ICD or N3FL for 24 h, followed by treatment with NAC (5 mM, 0-12 h). E. Subcellular analysis of Notch3 protein levels following NAC treatment (5 mM, 6 h) in HeLa cells. Protein levels of N3FL, N3EC and N3IC in cytosolic, nuclear and membrane fractions were determined. Successful fractionation was evidenced by using the marker proteins GAPDH, cyclin B1, and Na + , K + -ATPase. N3FL, N3EC and N3IC denoted Notch3 full length, extracellular domain and intracellular domain, respectively. Protein densitometry quantifications were shown after normalization with β-actin (A, C, D) or their respective cellular compartment markers (E). Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Transfection, Membrane, Fractionation, Marker

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: N3ICD overexpression rescues NAC-induced inhibition of proliferation (A), migration (B), and invasion (C). A. Numbers of EV- and N3ICD-transfected cells were counted at 12-48 h after NAC treatment (0-10 mM, left panel). *, p < 0.05 compared with the EV-transfected cells within the same treatment and time point. B. Results of the wound healing assay (left panels) were expressed as the migration index (the distance migrated relative to the initial scraped gap) and that of EV-transfected cells without NAC treatment was set as 100% (middle panel). C. Cells per field on the insert membrane were imaged (left panels) and counted (middle panel). B and C: *, p < 0.05 compared with no NAC treatment; #, p < 0.05 compared with the EV-transfected cells within the same treatment. Percent rescue (A-C, right panels) after N3ICD expression was calculated by dividing the net change after NAC treatment in N3ICD-transfected cells by that in EV-transfected cells. Notch3 siRNA knockdown inhibits cell proliferation D. , migration E. , and invasion F. as assessed by the same approaches described above. Representative images for migration and invasion were shown. *, p < 0.05 compared with the siCtrl-transfected cells. All data are presented as mean ±SE, n=3. I, the initial seeded cell number. EV, empty vector; N3ICD, Notch3 active intracellular domain; siCtrl, scrambled siRNA; siNotch3, Notch3 siRNA.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Over Expression, Inhibition, Migration, Transfection, Wound Healing Assay, Membrane, Expressing, Knockdown, Plasmid Preparation

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (5 and 10 mM, 0-24 h) decreases N3IC protein levels in HCC1937 cells. Expression of exogenous N3ICD rescues NAC-induced inhibition of proliferation B. , migration C. , and invasion D. , and Notch3 siRNA knockdown inhibits proliferation E. , migration F. , and invasion G. in HCC1937 cells. Quantifications, sample size, statistics, and abbreviations for protein levels, proliferation, migration, and invasion assays were as described in Figure & legends.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Inhibition, Migration, Knockdown

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 1 c-Cbl ubiquitin ligase regulates proteasomal degradation of Notch3-IC. (a) Co-transfection of HEK293 cells with flag Notch3-IC and HA-c-Cbl expression plasmids. Whole lysates were immunoprecipitated with anti-flag antibody and revealed in western blot with anti-Ub and anti-HA antibodies. Anti-flag antibody was used as a control of the immunoprecipitation assay. Total lysates were analyzed for the expression of c-Cbl plasmid using anti-HA antibody. (b, c) Co-transfection of HEK293 cells with flag Notch3-IC and HA c-Cbl expression plasmids: cells were treated with (b) proteasome inhibitor MG132 in a time course study for 0–5 h or with (c) cycloheximide (CHX) and/or MG132 for 5 h before lysis. In both experiments, whole lysates obtained were revealed in western blot with anti-flag antibody, followed by anti-b-actin antibody used as a control of sample loading (left panels). The right panels show the relative quantification as determined by optical densitometry (OD).

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Ubiquitin Proteomics, Cotransfection, Expressing, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Lysis

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 3 Protein kinase C y (PKCy) regulates c-Cbl phosphorylation status in ex vivo and in vitro experiments, resulting in the regulation of Notch3-IC protein levels. (a) Whole-cell extracts of thymocytes from wt, tg N3-IC and N3-IC/pTa/ mice were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P- Ser PKC substrate antibody. Anti-c-Cbl immunoblotting was used as a control of the immunoprecipitation assay. Western blot analysis of c-Cbl in total lysates shows that the modification of c-Cbl phosphorylation is not accounted for by changes of its protein levels. Data are representative of three similar experiments. (b) Treatment of N3-IC/pTa/ double-mutant thymocytes with phorbol 12-myristate 13-acetate (PMA) for 0–15–30 min before lysis: total lysates were revealed in western blot with anti-P-tyr and anti-Cbl antibodies. Molecular weight markers are shown on the left (left panel). The same samples were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P-Ser PKC substrate and anti-PKCy antibodies. Anti-c-Cbl immunoblotting was used as a control of the immunoprecipitation assay (right panel). (c) Treatment of N3-IC/ pTa/ double-mutant thymocytes with PMA for 3 h before lysis: western blot analysis of Notch3 protein levels. Anti-b-actin immunoblotting was used as a control of sample loading. (d) Co-transfection of HEK293 cells with HA-c-Cbl plasmid in presence or absence of DN-PKCy and CA-PKCy expression vectors. Whole lysates were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P-Ser and anti-PKCy antibodies. The immunoblotting with anti-Cbl antibody was used as a control of the immunoprecipitation assay. (e) Co-transfection of HEK293 cells with flag Notch3-IC and HA c-Cbl plasmids in presence or absence of DN-PKCy and CA-PKCy expression vectors. Whole lysates were immunoprecipitated with anti-flag antibody and revealed in western blot with anti-Ub antibody, followed by anti-flag antibody used as a control of the immunoprecipitation assay. The same whole lysates were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-flag antibody to detect c-Cbl/Notch3 interaction. (f) HEK293 cells, co-transfected as previously described, were treated with proteasome inhibitor MG132 for 5 h before lysis. Whole lysates were revealed in western blot with anti-flag antibody, followed by anti-b-actin antibody, used as a control of sample loading.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Phospho-proteomics, Ex Vivo, In Vitro, Immunoprecipitation, Western Blot, Control, Mutagenesis, Lysis, Molecular Weight, Cotransfection, Plasmid Preparation, Expressing, Transfection

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 4 pTa directly interacts with Notch3 and regulates its recruitment to lipid rafts. (a) Whole-cell extracts, cytosol and membrane fractions and rafts and nonrafts fractions of N3-232T cells were revealed in western blot with anti-N3EC and anti-N3IC antibodies. Results are representative of three similar experiments. (b) Left panel: confocal microscopy of thymocytes from wt and tg N3-IC mice stained with Alexa Fluor 594-conjugate CTB and rabbit polyclonal anti-Notch3 antibody, as described in Materials and methods section. Right panel: rafts and nonrafts fractions derived from the same cells were revealed in western blot with anti-N3EC and anti-pTa antibodies. Anti-ZAP-70 was used to control the activation status. For T-cell activation, cells were incubated with anti-CD3 antibody as described in Materials and methods section. (c) Rafts and nonrafts fractions derived from SCB29 and SCIET27 pre-T-cell lines were revealed in western blot with anti-N3EC antibody. (d) Whole-cell extracts (left panel), rafts and nonrafts fractions (right panel) derived from tg N3-IC and N3-IC/pTa/ mice were revealed in western blot with anti-N3EC. (e) Co-transfection of HEK293 cells with HA- Notch3-IC and pTa expression plasmids. Whole lysates were immunoprecipitated with anti-pTa antibody and revealed in western blot with anti-HA antibody, followed by anti-pTa antibody, used as a control of the immunoprecipitation assay. * indicates a nonspecific band (left panel). Whole lysates from 2017 pre-T-cell line were immunoprecipitated with anti-pTa antibody and revealed in western blot with anti-N3IC antibody, followed by anti-pTa antibody, used as a control of the immunoprecipitation assay (right panel). Data are representative of three similar experiments. Anti-p56Lck and anti-tubulin immunoblotting were used as a control of fractionation; anti-b-actin immunoblotting was used to monitor sample loading. WCE, whole-cell extracts; C, cytosol; M, membrane; R, rafts; NR, nonrafts; FL, full-length receptor; EC, extracellular fragment; TM, transmembrane fragment; IC, intracellular fragment; un, untransfected cells; tr, transfected cells.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Membrane, Western Blot, Confocal Microscopy, Staining, Derivative Assay, Control, Activation Assay, Incubation, Cotransfection, Expressing, Immunoprecipitation, Fractionation, Transfection

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 5 Notch3/pTa relationship favors c-Cbl recruitment to lipid rafts. (a) Rafts and nonrafts fractions from wt thymocytes, resting or activated with anti-CD3 antibody, and tg N3-IC thymocytes, not activated, were revealed in western blot with anti c-Cbl antibody. Anti-ZAP-70 and anti-b-actin immunoblotting were used to control the activation process and to monitor sample loading, respectively. (b) Treatment of N3-232T cells with furine-like inhibitor A23187 before rafts isolation. Rafts and nonrafts fractions were revealed in western blot with anti-c-Cbl antibody. Anti-N3EC and anti-N3IC immunoblotting were used as a control of the treatment; anti-p56Lck and anti-Lat immunoblotting were used as a control of rafts aggregation process. * indicates the Notch3-IC protein. (c) Rafts and nonrafts fractions from tg N3-IC and N3-IC/pTa/ thymocytes were revealed in western blot with anti-c-Cbl antibody. Anti-p56Lck and anti-tubulin immunoblotting were used as a control of fractionation. The samples are normalized using b-actin protein expression (left panel). The right panel shows the quantification of c-Cbl protein associated to rafts in tg N3-IC and N3-IC/pTa/ thymocytes. Data represent the average of three independent experiments. (d) c-Cbl/Notch3-IC cytosolic interaction after rafts disrupting. Treatment of N3-232T cells with methyl-b-cyclodextrin (MbCD) before subcellular fractionation: cytosolic fractions derived from N3- 232T MbCD-treated and -untreated cells were used for immunoprecipitation assay with anti-c-Cbl antibody and revealed in western blot with anti-N3IC antibody, followed by anti-c-Cbl antibody, used as a control of the immunoprecipitation assay. R, rafts; I, intermediate fractions; NR, nonrafts; FL, full-length receptor; EC, extracellular fragment; TM, transmembrane fragment.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Western Blot, Control, Activation Assay, Isolation, Fractionation, Expressing, Derivative Assay, Immunoprecipitation

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 6 Hypothetical model linking c-Cbl different subcellular localization with Notch3 degradation. (a) In presence of a functional pre-TCR/Notch3 relationship, c-Cbl is able to translocate to the rafts compartment, where it is preferentially phosphorylated in serine- rich motifs through protein kinase C y (PKCy). In these conditions c-Cbl seems to lack its E3 ubiquitin ligase upon Notch3, thus sustaining the oncogenic role of Notch3 and pTa relationship with respect to T-cell leukemogenesis. (b) In the absence of pre-TCR, c-Cbl localizes preferentially in cytosolic fraction. In addition, deletion of pTa results in reduction of PKCy activity that in turn favors tyrosine phosphorylation of c-Cbl and its increased E3 ubiquitin ligase activity upon Notch3-IC, targeting it to proteasomal-degradative pathway and contributing to the rescue of T-cell leukemia. P-tyr, tyrosine phosphorylation; P-Ser, serine phosphorylation; Ub, ubiquitination.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Functional Assay, Ubiquitin Proteomics, Activity Assay, Phospho-proteomics

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: eBioMedicine

Article Title: Chronic lung inflammation and CK14+ basal cell proliferation induce persistent alveolar-bronchiolization in SARS-CoV-2-infected hamsters

doi: 10.1016/j.ebiom.2024.105363

Figure Lengend Snippet: Highly elevated N otch signaling long after SARS-CoV-2 infection in hamster lung . a. Relative mRNA expression levels of Notch signaling related genes in the lung tissues in SARS-CoV-2 and IAV infected hamsters. n = 5 for IAV 42&120dpi, 6 for SARS-CoV-2 42dpi, 4 for SARS-CoV-2 120dpi. Data represents mean ± SD. b. Representative images of immunofluorescence stained Notch3 (green) in mock and SARS-CoV-2 infected hamster lung at 7, 14, 42, 84 and 120dpi. Scale bar = 100 μm or 20 μm. White triangles indicate Notch3 positive cells. White arrows indicate Notch3 signals inside nuclei. c. Representative magnified images of immunofluorescence stained Notch3 (green) captured by confocal microscopy in SARS-CoV-2 infected hamster lung at 14, 42, 84 and 120dpi. Scale bar = 5 μm or 10 μm. White arrows indicate Notch3 signals inside nuclei. d. Representative images of immunofluorescence stained Hes1 (green) in mock and SARS-CoV-2 infected hamster lung at 42 and 120dpi. Scale bar = 100 μm, 50 μm or 20 μm. White arrows indicate Notch3 positive cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by Two-way ANOVA with Tukey's multiple comparisons test (a).

Article Snippet: Viral antigens and cellular proteins were detected by immunohistochemistry or immunofluorescence with specific antibodies: home-made rabbit/mouse anti-SARS-CoV-2 nucleocapsid protein antibody, home-made mouse anti-H1N1 nucleocapsid protein antibody, anti-Ki67 antibody (556003, BD; ab15580, Abcam), anti-β tubulin-IV antibody (T7941, Sigma), anti-CK14 antibody (PA516722, Invitrogen; MA5-11599, Invitrogen), anti-SCGB1A1 antibody (10490, Proteintec), anti-SPC antibody (ab3786, Millipore), anti-Iba1 antibody (ab178846, Abcam), anti-Notch3 antibody (MBS242006, MyBioSource), anti-Hes1 antibody (LS-B2211 LSBio), anti-Sox2 antibody (11064-1-AP, Proteintech) and anti-Mlf1 antibody (MBS9605647, MyBioSource).

Techniques: Infection, Expressing, Immunofluorescence, Staining, Confocal Microscopy