Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

doi: 10.1038/labinvest.2015.149

Figure Lengend Snippet: Figure 3 Notch signaling pathway was activated in CLF. (a) Notch-1, 2, 3, 4 mRNA. (b) JAG1, 2, and DLL1, 3, 4 mRNA. (c) Hes1, Numb, and RBPJк mRNA. All mRNA were quantified with RT-PCR and normalized to GAPDH mRNA (n = 6 per group). (d) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb protein expression were quantified via immunoblotting and normalized to GAPDH (n = 6 per group), and (e) densitometric analysis of protein bands. *Po0.05, **Po0.01. Sham, Sham group; 1 wM, BDL-1w group; 2 wM, BDL-2w group; 3 wM, BDL-3w group; 4 wM, BDL-4w group.

Article Snippet: Rabbit polyclonal antibodies to Notch4 (sc-5594), JAG1 (sc-8303), DLL1 (sc-9102), mouse monoclonal antibody to Sox9 (E-9, sc-166505), and goat polyclonal antibody to JAG2 (sc-34475) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

doi: 10.1038/labinvest.2015.149

Figure Lengend Snippet: Figure 6 Inhibition of the Notch signaling pathway reduced the progression of liver fibrosis. (a) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb, protein bands on the left (immunoblot), and the histogram depicts the densitometric analysis of protein bands (n = 6 per group). (b) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb mRNA were measured via RT-PCR and normalized to GAPDH mRNA (n = 6 per group). (c) Sirius Red staining (×100). (d) α-SMA immunostaining (×200). (e) Hydroxyproline content. (f) α-SMA, TNF-α, TGF-β1, MCP-1, Col(1), and Col (4) mRNA were measured by RT-PCR and normalized to GAPDH mRNA (n = 6 per group). *Po0.05, **Po0.01. BDL, single bile duct ligation group; DAPT, BDL plus DAPT group; sham, sham group.

Article Snippet: Rabbit polyclonal antibodies to Notch4 (sc-5594), JAG1 (sc-8303), DLL1 (sc-9102), mouse monoclonal antibody to Sox9 (E-9, sc-166505), and goat polyclonal antibody to JAG2 (sc-34475) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Immunostaining, Ligation

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Abnormal expression of NOTCH1–4. (A) Full landscape of NOTCH1–4 expression profiles in multiple cancer types in the TIMER database. (B) TCGA-based UALCAN database screening and analysis showing a significant difference in the NOTCH1 and NOTCH4 expressions between normal tissue and PCa tissue (P=1.047e-6, 1.05e-1, 5.70e-2, 5.93e-5, respectively). (C) IHC staining of NOTCH1–4 in PCa or BPH tissues respectively [N (tumor) =15, N (BPH) =6]. (D) qRT-PCR results for the mRNA levels of PCa tissues and adjacent normal prostate tissues. (T =24, N =24). *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001. ns, no significant. PRAD, prostate adenocarcinoma. qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; BPH, benign prostate hyperplasia; IHC, immunohistochemistry; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Correlation between NOTCH1–4 expression and Gleason score or prognosis in PCa patients. (A) UALCAN database analysis showing that the transcriptional level of NOTCH3 and NOTCH4 is significantly enhanced with increasing Gleason score. (B) The Mantel–Cox test with GEPIA2 showing that the expression of NOTCH3 and NOTCH4 exhibited a positive correlation with prognosis in PCa patients. (C) Cox regression analysis showing that patients in the low NOTCH1, 3 and 4 groups had a significant better disease-free survival (DFS) than those in the high group (P=4.8e-2, 5.6e-3, and 2.0e-2, respectively). (D) Cox regression analysis showing that there was no significant difference in overall survival (OS) between high and low NOTCH family genes’ expression. *, P<0.05, **, P<0.01, ***, P<0.001 and ****, P<0.0001. GS, Gleason score; PRAD, prostate adenocarcinoma; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Neighbor gene network and interaction analyses of NOTCH1–4 in PCa patients. (A) Correlation heat map of NOTCH family genes and NOTCH-related genes in PCa patients. (B,C) Protein-protein interaction networks of differently expressed NOTCH family genes. PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques:

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Characterization of genetic alterations and mutations of NOTCH1–4. (A) Mutational frequencies of NOTCH1, NOTCH2, NOTCH3 and NOTCH4 in PCa were 7%, 8%, 5%, and 5%, respectively. (B) Top 10 most significant mutation-related genes of NOTCH1–4. (C) Wayne chart showing the shared mutation-related genes of NOTCH1–4. (D) Bar graph showing the top 20 results from the GO enrichment analysis. (E) GO enrichment analysis showing the gene networks. GO, gene ontology; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Mutagenesis

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Common differentially expressed genes among NOTCH1–4 mutations

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Derivative Assay

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: NOTCH1–4 mutations and drug sensitivity. (A) Volcano plots showing that multiple cancer cell types with the NOTCH1 mutation were sensitively targeted by BMS-754807, linsitinib, saracatinib and erlotinib (left-hand panel). Tumor cells with the NOTCH2 mutation showed potential antagonism against entospletinib (right-hand panel). (B) Scatter plots showing that tumor cells were significantly suppressed by the use of BMS-754807, linsitinib, saracatinib, erlotinib and entospletinib in the NOTCH1 mutation group compared with the wild-type group (P<0.05 for all). *, P<0.05, **, P<0.01, and ***, P<0.001. IC50, half maximal inhibitory concentration; GDSC, genomics of drug sensitivity in cancer; Mut, mutation.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Mutagenesis, Concentration Assay

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: NOTCH mutation-related drug-sensitivity analyzed using ANOVA

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Mutagenesis

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Correlation between differently expressed NOTCH1–4 and lymphatic metastasis or immune cell infiltration. (A) TIMER database analysis showing that patients with N1 stage had significant higher expression of NOTCH3 than those with N0 stage (P<0.05, N0 = no lymphatic metastasis, N1 = lymphatic metastasis). (B) Associations between NOTCH1–4 and lymphocytes infiltration in PCa patients. *, P<0.05, ***, P<0.001 and ****, P<0.0001. ns, no significant. PCa, prostate cancer; PRAD, prostate adenocarcinoma; TIMER, tumor immune estimation resource.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Fringe GlcNAc-transferases differentially extend O -fucose on endogenous NOTCH1 in mouse activated T cells

doi: 10.1016/j.jbc.2022.102064

Figure Lengend Snippet: Endogenous N1 from preT 2017 cells was modified by O -fucose at high stoichiometry, but only the EGF16 O- fucose was extended by Fringes. A , summary of O -fucose modifications identified by mass spectral analysis of endogenous mN1 isolated from preT 2017 cells. Most extended form of the O -fucose glycan detected is shown. Mass spectral data in Fig. S10 and Table S2 . B , extracted ion chromatogram showing relative amounts of O -fucose peptide glycoforms from EGF12 and EGF16. Black , red , and blue lines indicate the unmodified, O -fucose monosaccharide, and both O -fucose and O -glucose monosaccharide glycoforms in the EGF12 panel, respectively. Black , red , blue , green , and magenta lines indicate the unmodified, monosaccharide, disaccharide, trisaccharide, and tetrasaccharide O -fucose glycoforms in the EGF16 panel, respectively. C , cell surface N1 expression measured by flow cytometry in preT 2017 cells or in HEK293T cells overexpressing (O/E) mN1 or empty vector (EV) control. Note that the mouse N1 antibody used here has slight crossreactivity with human N1. D , Western blot analysis of endogenous mN1 in preT 2017 lysate or overexpressed mN1 from HEK293T lysate with anti-N1 ECD antibody. ECD, extracellular domain; EGF, epidermal growth factor-like.

Article Snippet: For cell surface mN1 detection, washed cells were incubated with sheep anti-mN1 antibody (AF5267; R&D Systems, 1:1000 dilution) or sheep nonspecific IgG (R&D Systems, 1:1000 dilution) on ice for 1 h. N1 antibody or nonspecific IgG were detected by antisheep Fc PE conjugate antibody (Thermo, 1/25).

Techniques: Modification, Isolation, Glycoproteomics, Expressing, Flow Cytometry, Plasmid Preparation, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Fringe GlcNAc-transferases differentially extend O -fucose on endogenous NOTCH1 in mouse activated T cells

doi: 10.1016/j.jbc.2022.102064

Figure Lengend Snippet: EGF12 is not modified by Fringe in Fng tKO activated T cells. A , EICs show relative levels of mN1 control peptide lacking an O- fucose site from EGF12 ( black line : 475 QCICMPGYEGVY 486 ) versus mN1 EGF12 peptide with monosaccharide O -fucose modification ( red line : 445 TGPRCEIDVNECISNPCQNDA T CLDQIGEF 474 , O -fucose site bold underlined) from Fng LMR or Fng tKO activated T cells. B , ratio of EGF12 peptide to control peptide from mN1 expressed in HEK293T cells in the absence or presence of LFNG (shown in Fig. S9 ) or mN1 isolated from Fng LMR or Fng tKO activated T cells. NS ≥ 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Mass spectral data for EGF12 and control peptide are shown in Table S4 . Error bars reflect ± SD. EGF, epidermal growth factor-like; EIC, extracted ion chromatogram; tKO, triple knockout.

Article Snippet: For cell surface mN1 detection, washed cells were incubated with sheep anti-mN1 antibody (AF5267; R&D Systems, 1:1000 dilution) or sheep nonspecific IgG (R&D Systems, 1:1000 dilution) on ice for 1 h. N1 antibody or nonspecific IgG were detected by antisheep Fc PE conjugate antibody (Thermo, 1/25).

Techniques: Modification, Control, Isolation, Triple Knockout

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