Journal: Nagoya Journal of Medical Science

Article Title: XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting cell proliferation

doi: 10.18999/nagjms.87.3.431

Figure Lengend Snippet: NOTCH1 EGF repeats overexpressed in Jurkat cells are modified with the O- glucose trisaccharide at EGF10 Fig. 1A: HCD-MS/MS spectra of a glycopeptide from NOTCH1EGF10 carrying the O- Glc trisaccharide Xyl-Xyl-Glc. Numerous b and y ions in addition to the sequential neutral loss of sugar residues from the precursor ion confirm the identity of the peptide and the presence of the O- Glc trisaccharide. Fig. 1B–E: EICs of glycopeptides from EGF10 modified with previously reported glycoforms. Blue circles, glucose; Orange stars, Xyl; White circles, uncharacterized hexose; Black dashed line, naked peptide; Blue, peptide + O- Glc; Yellow, peptide + O- Glc-Xyl; Orange, peptide + O- Glc-Xyl-Xyl; Gray, peptide + 2 hexoses; Purple, peptide + 2 hexoses and sialic acid. (B) WT Jurkat cells, (C) Cas9 control cells, (D) XXYLT1 KO #1 (gRNA-3 transfectant), and (E) XXYLT1 KO #3 (gRNA-1 transfectant). EGF: epidermal growth factor-like HCD: higher-energy collision dissociation MS/MS: tandem mass spectrometry Glc: glucose Xyl: xylose EICs: extracted ion chromatograms WT: wild-type Cas9: CRISPR-associated protein 9 XXYLT1: xyloside α1-3xylosyltransferase 1 KO: knockout gRNA: guide RNA

Article Snippet: Total cell lysates (5 μg) were loaded and subsequently analyzed by western blotting using anti-αTubulin antibody (DSHB, 12G10, 1:4000), anti-cleaved NOTCH1 (Val1744) antibody (CST, D3B8, 1:1000), and anti-HA antibody (MBL, 561, 1:1000), and the intensity of cleaved NOTCH1 was normalized to the intensity of αTubulin.

Techniques: Modification, Tandem Mass Spectroscopy, Glycoproteomics, Control, Transfection, Mass Spectrometry, CRISPR, Knock-Out

Journal: Nagoya Journal of Medical Science

Article Title: XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting cell proliferation

doi: 10.18999/nagjms.87.3.431

Figure Lengend Snippet: The activation of NOTCH1 is enhanced in XXYLT1 KO Jurkat clones Fig. 2A: Western Blot analysis of WT, Cas9-control, and XXYLT1 KO clones using antibodies against activated NOTCH1 (Val1744) and α-tubulin (loading control). Fig. 2B: Normalized intensities of activated NOTCH1 signals from five independent experiments. Error bars indicate standard deviation. Significance was calculated using a one-way ANOVA followed by Dunnett’s test. Fig. 2C: Western Blot analysis of NOTCH1 activation in empty vector or XXYLT1-HA -transduced WT and XXYLT1 KO cells. Fig. 2D: Normalized intensities of activated NOTCH1 signals from three independent experiments. Error bars indicate standard deviation. Significance was calculated using a one-way ANOVA followed by Tukey’s test. Fig. 2E: Flow cytometry analysis of cell surface NOTCH1 in WT, Cas9-control, and XXYLT1 KO clones. Normalized mean fluorescence intensities from four independent experiments were plotted. Error bars represent the standard deviation. Significance was calculated using a one-way ANOVA followed by Dunnett’s test. XXYLT1: xyloside α1-3xylosyltransferase 1 KO: knockout WT: wild-type Cas9: CRISPR-associated protein 9 ANOVA: analysis of variance HA: hemagglutinin tag MFI: mean fluorescence intensity

Article Snippet: Total cell lysates (5 μg) were loaded and subsequently analyzed by western blotting using anti-αTubulin antibody (DSHB, 12G10, 1:4000), anti-cleaved NOTCH1 (Val1744) antibody (CST, D3B8, 1:1000), and anti-HA antibody (MBL, 561, 1:1000), and the intensity of cleaved NOTCH1 was normalized to the intensity of αTubulin.

Techniques: Activation Assay, Clone Assay, Western Blot, Control, Standard Deviation, Plasmid Preparation, Flow Cytometry, Fluorescence, Knock-Out, CRISPR

Journal: Cancer Research

Article Title: Aberrant Activation of Notch Signaling in Human Breast Cancer

doi: 10.1158/0008-5472.can-05-3054

Figure Lengend Snippet: Figure 1. Expression of Notch receptors and DSL ligands in the normal human breast. A-C, expression of Notch1 and Notch3 (lanes A2 and A4), Delta-like 4 (lane B4), and Jagged1 and Jagged2 (lanes C2 and C3) was detected in normal human breast tissue by reverse transcription-PCR (+RT, top). In contrast, primers specific to Notch2 (lane A3), Notch4 (lane A5), Delta-like 1 (lane B2), and Delta-like 3 (lane B3) genes failed to amplify a fragment, indicating that these genes were not expressed (+RT, top). Control reactions without reverse transcriptase were set up in parallel (RT, bottom). N, Notch; Dll, Delta-like; Jag, Jagged. D, immunolocalization of Notch receptors and DSL ligands identified by reverse transcription-PCR analysis of normal breast epithelial cell lines and tissue samples. Sections of normal human breast tissue were stained with antibodies that recognize Notch1, Notch3, Jagged1, and Jagged2. All four proteins were observed in the lobular epithelium and not in surrounding adipose tissue (arrows). Adjacent sections were stained without primary anti-Notch1, anti-Notch3, anti-Jagged1, and anti-Jagged2 antibodies as controls. Samples were counterstained with haemotoxylin to reveal morphology.

Article Snippet: Primary antibodies used were as follows: anti-cleaved Notch1 (Rockland), anti-Numb (Abcam, Cambridge, MA), anti-Noxa (Abcam), antiPuma (Novus Biologicals, Littleton, CO), anti-cleaved Caspase3 (Cell Signaling Technology, Beverly, MA), anti-Desmoplakin (kind gift of Prof. David Garrod, University of Manchester), anti-Hey1 (Santa Cruz Biotechnology), anti-E-cadherin (BD Biosciences, San Jose, CA), antitubulin (kind gift of Prof. Keith Gull, University of Oxford), anti–phosphoc-Jun NH2-terminal kinase (JNK) pT 183pY185 (Biosource, Camarillo, CA), anti–phopho-p53 pT81 (Cell Signaling Technology), anti-Keratin18 (NeoMarkers, Fremont, CA), anti-Actin (Sigma), and anti-focal adhesion kinase Cancer Res 2006; 66: (3).

Techniques: Expressing, Reverse Transcription, Control, Staining

Journal: Cancer Research

Article Title: Aberrant Activation of Notch Signaling in Human Breast Cancer

doi: 10.1158/0008-5472.can-05-3054

Figure Lengend Snippet: Figure 4. Notch signaling is elevated in human breast cancer cell lines and tissue samples. A, Western blot analysis of NICD, Numb, and Hey1 levels in three normal (lanes 1-3) and eight tumorigenic human breast epithelial cell lines (lanes 4-11). Numb expression was undetectable in all eight cancer cell lines tested, whereas NICD and Hey1 were up-regulated. The blot was stripped and reprobed with an antibody that recognizes actin to confirm equal loading. B, Western blot analysis of NICD and Numb levels in two normal and nine breast cancer tissue samples. Protein lysates from two normal tissue samples (lanes A and B) were separated by SDS-PAGE with either seven ER and PR expressing tumors (tumors C1-C7; Table 1), seven ER expressing tumors that lack PR (tumors D1-D7, Table 1), or six tumors that overexpress ErbB2 or EGFR (tumors E1-E3, F1, and G1 and G2). Representative examples from these blots are shown along with the two normal samples. As with the cancer cell lines, Numb was down-regulated in all breast cancer patients, whereas NICD was up-regulated. The blots were stripped and reprobed with an antibody that recognises epithelial marker Desmoplakin to confirm equal loading of the epithelial compartment. C, immunolocalisation of NICD, Numb, and Muc1 in a representative breast cancer tissue sample. Adjacent sections were stained with antibodies that recognise the NICD, Numb, and Muc1 (bottom). Down-regulation of Numb was accompanied by up-regulation of the active form of Notch in the epithelial cells as judged by Muc1 expression. Adjacent sections were stained without primary anti-Notch1, anti-Numb, and anti-Muc1 antibodies as controls (top). Samples were counterstained with hematoxylin to reveal morphology.

Article Snippet: Primary antibodies used were as follows: anti-cleaved Notch1 (Rockland), anti-Numb (Abcam, Cambridge, MA), anti-Noxa (Abcam), antiPuma (Novus Biologicals, Littleton, CO), anti-cleaved Caspase3 (Cell Signaling Technology, Beverly, MA), anti-Desmoplakin (kind gift of Prof. David Garrod, University of Manchester), anti-Hey1 (Santa Cruz Biotechnology), anti-E-cadherin (BD Biosciences, San Jose, CA), antitubulin (kind gift of Prof. Keith Gull, University of Oxford), anti–phosphoc-Jun NH2-terminal kinase (JNK) pT 183pY185 (Biosource, Camarillo, CA), anti–phopho-p53 pT81 (Cell Signaling Technology), anti-Keratin18 (NeoMarkers, Fremont, CA), anti-Actin (Sigma), and anti-focal adhesion kinase Cancer Res 2006; 66: (3).

Techniques: Western Blot, Expressing, SDS Page, Marker, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Inhibition of the Notch pathway aggravates DSS-induced colitis. CT: control, LY411,575 (Notch inhibitor): 10 mg/kg LY411,575 gavage, DSS: 2.5% DSS water, DSS-LY411,575: LY411,575 was intragastric administrated at the last 4 days of DSS feeding (once per day). (A) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (B) Serum FD-4 concentration in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (C) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (D) Representative colonic H&E staining image of each group (scale bar = 50 μm). (E) Representative colonic IF staining image of claudin-1 ( green ) and claudin-3 ( green ) (scale bar = 25 μm). (F) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) (scale bar = 25 μm). Quantification of N1ICD and Hes1 were determined by the ratio of positive staining cells per crypt. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Inhibition, Control, Concentration Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Notch pathway is inhibited in the colon under VDd or VDR KO conditions. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO (A) and VDd (B) mice (scale bar = 25 μm). (C, D) Real-time PCR analysis of the gene expression of Notch ligands, receptors, and effectors in the colon in VDR KO (C) and VDd (D) mice. Expression levels (VDR KO vs. WT or VDd vs. CT) were compared using paired t -test. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR modulates the Notch pathway in colitis. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDd-DSS and respective control colitis mice (scale bar = 25 μm). (C, D) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO-DSS and respective control colitis mice. To activate VDR, mice were administered paricalcitol (PAR) 7 days before drinking DSS water and were sustained until sacrifice. (E) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (F) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (G) Representative colonic H&E staining image of each group (scale bar = 50 μm). (H) Representative colonic IF staining image of claudin-1 ( green ), claudin-3 ( green ), and Hes1 ( red ) (scale bar = 25 μm). Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling regulates the Notch pathway and TJs in Caco-2 cells. TNF-α (100 ng/mL) was added to mimic inflammatory stimulation, and 100 nM PAR was used to activate VDR; the solvate was added as control (CT). Specific siRNA of VDR (SiVDR) was used to downregulate VDR, negative control (NC) sequence was used as control (scale bar = 10 μm). (A) Representative IF staining image of claudin-1 ( green ) and claudin-3 ( green ) in Caco-2 cells treated with or without PAR before TNF-α intervention. (B) Representative IF staining image of N1ICD ( red ) and Hes1 ( red ) in Caco-2 cells with or without VDR downregulation before TNF-α intervention. (C) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in Caco-2 cells after VDR downregulation.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Negative Control, Sequencing, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR-mediated TJ protection is partly dependent on the Notch pathway. LY411,575 (1 μM) was used to inhibit the Notch pathway in Caco-2 cells, and 100 nM PAR was used to activate VDR. The solvate was added as control (CT). Representative IF staining image of N1ICD ( red ), Hes1 ( red ), claudin-1 ( green ), and claudin-3 ( green ) in Caco-2 cells treated with or without LY411,575 before PAR intervention (scale bar = 10 μm).

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling positively regulates the Notch-1/Hes1 pathway. (A) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in cultured intestinal organoids (scale bar = 10 μm). (B) Western blot analysis of N1ICD and Hes1 proteins in cultured intestinal organoids from WT and VDR KO mice. (C) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in cultured intestinal organoids from WT and VDR KO mice. (D) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after TNF-α intervention. (E, F) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after downregulation of VDR with siVDR (E) or activation of VDR with PAR (F) . Data are presented as the mean ± SEM from three separate experiments, the statistical analysis performed with paired t -test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR positively regulates Notch-1 transcription. (A) Western blot analysis of VDR, Notch-1, N1ICD, and Hes1 in Caco-2 cells after siVDR and PAR intervention. (B) Graphs of the predicted VDR binding region in the Notch-1 promoter. (C) Dual-luciferase reporter assay of VDR to Notch-1 promoter. Notch-1 promoter (Notch-1) and negative control (CON) were constructed in a GV238 vector. The VDR cDNA and vector (pcDNA3.1) were linked to upregulate VDR (pVDR). Data are presented as the mean ± SEM from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Western Blot, Binding Assay, Luciferase, Reporter Assay, Negative Control, Construct, Plasmid Preparation