Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 2: Overexpression of UGDH in the AD LNCaP background desensitizes AR-mediated gene expression and reduces glucuronidation precursors. Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP AD background. Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells were harvested for analysis. AR-dependent genes PSA (A) and UGT2B17 (B) were analyzed by WB in whole cell lysates; (C) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; (D) UDP-sugar pools were measured in cell lysates by LC-MS. In panels A–C, mean ± SEM is plotted for triplicate measurements. In panel D, mean ± SD is plotted for quadruplicate measurements. Statistical significance is indicated as: (a) p < 0.05 relative to VC1 at 0 nM DHT. (b) p < 0.05 relative to VC2 at 0 nM DHT. (c) p < 0.05 comparing OE1 to both VC1 and VC2 at the indicated [DHT]. (d) p < 0.05 comparing OE2 to both VC1 and VC2 at the indicated [DHT].

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Over Expression, Gene Expression, Plasmid Preparation, Control, Concentration Assay, Functional Assay, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 3: Overexpression of UGDH in the CR LNCaP background further suppresses AR-mediated expression of glucuronidation genes and reduces nucleotide sugar pools without impacting proteoglycan production. Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP CR background. Equal cell numbers were seeded 48 hours in androgen replete media followed by harvest of cells for analysis of gene expression (A and B) and UDP-sugars (D). For panel (C), equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells and media were harvested for analysis. AR-dependent genes UGT2B17 (A) and FoxA1 (B) were analyzed by WB in whole cell lysates; (C) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; (D) UDP-sugar pools were measured in cell lysates by mass spectrometry. Mean ± SEM is plotted for triplicate technical measurements; *p < 0.05 for OE1 and OE2 relative to VC1 and VC2.

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Gene Expression, Concentration Assay, Functional Assay, Mass Spectrometry

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 5: Loss of UGDH promotes AR-dependent gene expression and reduces proteoglycan production while sustaining glucuronide output. LNCaP AD and CR cells were selected for stable expression of a non-targeting vector control (VC1 and VC2) or a UGDH shRNA knockdown construct (KD1 and KD2). Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing 10 nM DHT or DMSO (vehicle, 0 nM). After an additional 48 hours, cells were harvested for analysis by western blot and mass spectrometry. (A) AR-dependent genes PSA (panels a and d) and UGT2B17 (panels b and e) were analyzed by WB in whole cell lysates; functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates (panels c and f). Panels (a–c) depict expression data in the AD background and panels (d–f) illustrate data from the CR background. Mean ± SEM is plotted. Statistical significance is indicated on the plots as: (a) p < 0.05 for that clone, comparing 0 vs 10 nM DHT; (b) p < 0.05 relative to VC1 and VC2, 10 nM DHT; (c) p < 0.05 relative to VC1 and VC2, 0 nM DHT. (B) UDP-sugar pools were measured in cell lysates by LC-MS for both the AD (upper) and CR (lower) backgrounds as indicated. Mean ± SD is plotted. Statistical significance is indicated as: (a) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 0 nM DHT. (b) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 10 nM DHT. (c) p < 0.05 for that clone, comparing 0 and 10 nM DHT.

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Gene Expression, Expressing, Plasmid Preparation, Control, shRNA, Knockdown, Construct, Western Blot, Mass Spectrometry, Functional Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 4 (a) Selected gene expressions regarding epithelial mesenchymal markers, differentiation makers and regulators from RNA- sequencing data. (b and c) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day4 after siRNA. *p < 0.05. Data presented are from one of two independent experiments with similar results. (d and e) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR. *p < 0.05. Data presented are from one of two independent experiments with similar results. (f) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with siRNA and 3D organoid matrigel. *p < 0.05. Data presented are from one of two independent experiments with similar results. (g) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Expressing, Transfection

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 5 TINCR binds to STAU1 protein and controls critical regulators of differentiation. (a) Bar graphs show percentage of TINCR in the cytoplasm (black) and nucleus (white). NEAT1 serves as a positive control for nucleus enriched RNA and GAPDH serves as a positive control for cytoplasmic RNA. Data presented are from two independent experiments. (b) RIP experiments were performed using isotype IgG and STAU1 antibody to immunoprecipitated STAU1 protein/mRNAs complexes in total-cell extracts of NHBECs, and relative enrichment was determined as RNA associated with STAU1 IP relative to an input control. Relative ARF1 enrichment served as a positive control and NEAT1 as a negative control as NEAT1 does not interact with STAU1. Data presented are from two independent experiments. (c) Relative mRNA enrichment of STAU1 antibody in total-cell extracts of NHBECs transfected with siSCR or siTICNR. Data presented are from two independent experiments. (d and e) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU1. NHBECs were seeded on 6 well plate at 2 × 105 density and analyzed at day4 after transfection of siRNA reagents. *p < 0.05. (f and g) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU after transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR for 4 h. NHBECs were seeded on 24 well plate at 1 × 105 density and analyzed at day4 after transfection of siRNA reagents

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: Positive Control, Immunoprecipitation, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 1. Notch1-shRNA suppresses L02/HBx cell proliferation in vitro. (A) Identification of the effective shRNA targeting the Notch1 gene. The relative mRNA and protein levels of Notch1 were assessed by qRT-PCR and western blotting in L02/HBx cells 48 h after transient transfection with Notch1-shRNA1, Notch1-shRNA2 or negative control-shRNA (NC), respectively. (B) The components of the Notch1 signaling pathway were downregulated in the L02/ HBx-Notch1 shRNA2 cells. The mRNA and protein expression levels of Notch1 and Hes1 were assessed by qRT-PCR and western blotting, respectively. Actin was used as a loading control for both quantitative RT-PCR and western blotting. (C) CCK-8 assay and (D) colony formation assay of L02/HBx cells stably transfected with control or Notch1 shRNA2. Data are shown as the mean ± SEM from at least three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, In Vitro, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Expressing, Control, CCK-8 Assay, Colony Assay, Stable Transfection

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 2. Notch1-shRNAs suppresses L02/HBx cell proliferation in vivo. (A) The growth curve of the tumors derived from L02/HBx cells pretreated with Notch1-shRNA or control-shRNA in nude mice. Data represent means ± SEM of six samples. *P<0.05, **P<0.01. (B) Images of representative mice and dis sected tumors from nude mice. (C) Tumor tissues from nude mice inoculated with NC or Notch1-shRNA cells were stained with hematoxylin and eosin (H&E). Original magnification, x200. (D) Immunohistochemistry of Notch1 expression in tumor tissues from nude mice. Original magnification, x400.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: In Vivo, Derivative Assay, shRNA, Control, Staining, Immunohistochemistry, Expressing

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 3. Notch1-shRNA induces cell cycle arrest via the CyclinD1/CDK4 pathway in L02/HBx cells. (A) Notch1-shRNA induced cell cycle arrest in L02/ HBx cells. Cell cycle distribution was examined by flow cytometry after staining with PI. Results are visualized as a representative experiment or means ± SEM of three experiments. (B) Notch1-shRNA downregulates the CyclinD1/CDK4 pathway in L02/HBx cells. The expression levels of cell cycle regulatory genes CyclinD1, CDK4, E2F1, Rb, p21 and CyclinE1 were analyzed by qRT-PCR and western blotting. Data represent the mean ± SEM from three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, Cytometry, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 4. Notch1-shRNA increases cell apoptosis via the caspase-9-caspase-3 pathway in L02/HBx cells. (A) Notch1-shRNA increased cell apoptosis in L02/ HBx cells. Analysis of apoptosis was performed using PE Annexin V and 7-AAD staining by FACS analysis. Results are visualized as a representative experi ment or means ± SEM of three experiments. (B) Notch1-shRNA upregulated the caspase-9-caspase-3 pathway in L02/HBx cells. The expression of caspase-9, -8 and -3 was analyzed by qRT-PCR and western blotting. Data are shown as the mean ± SEM of three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncology Letters

Article Title: Functional redundancy of the Notch pathway in ovarian cancer cell lines

doi: 10.3892/ol.2016.4959

Figure Lengend Snippet: Vorinostat increases the mRNA expression of Notch receptors, Dll/Jagged ligands and Hey/Hes downstream target genes. Ovarian clear cell carcinoma ES2 cells were grown in the absence and presence of vorinostat (5 µM) for 6, 18 and 30 h, following starvation, with medium supplemented with 1% fetal bovine serum. (A) Notch receptor expression levels in ES2 cells. RT-qPCR revealed that Notch2, Notch3 and Notch4 mRNA expression was increased following vorinostat exposure, whereas Notch1 was not differentially expressed. (B) Representative staining of Notch receptors by immunofluorescence. It was observed that there was an increase of Notch2 and Notch4 protein following exposure to vorinostat (magnification, ×200). Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue) and Notch receptors with fluorescein isothiocyanate (green). (C) Notch ligand expression levels in ES2 cells. RT-qPCR indicated that Dll1, Dll3, Dll4 mRNA expression levels in cells treated with vorinostat were increased compared with cells without vorinostat treatment (magnification, ×200). Jagged1 and Jagged2 mRNA expression levels were not significantly different. (D) Notch downstream target gene expression levels. RT-qPCR revealed that Hes1, Hes5, Hey1 and Hey2 mRNA expression levels in cells treated with vorinostat were increased compared with cells without vorinostat treatment. Hes6 had a similar expression under all conditions. RT-qPCR was normalized to the hypoxanthine phosphoribosyltransferase gene. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05; **P<0.01; ***P≤0.001 vs. control cells. Dll, Delta-like; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Hes, hairy enhancer of split; Hey, Hes-related proteins.

Article Snippet: The primary antibodies were as follows: Rabbit polyclonal anti-human Notch1 extracellular (catalog no., ABS90; EMD Millipore, Billerica, MA, USA), rabbit monoclonal anti-Notch1 cleaved (catalog no., 4147; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-Notch2 cleaved (catalog no., 07-1234; EMD Millipore) and rabbit polyclonal anti-Notch4 (catalog no., N5163; Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Targeted Gene Expression, Standard Deviation, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Functional redundancy of the Notch pathway in ovarian cancer cell lines

doi: 10.3892/ol.2016.4959

Figure Lengend Snippet: Vorinostat increases the mRNA expression of Notch receptors, ligands and downstream targets in ovarian serous carcinoma OVCAR3 cell line. The cells were grown in the absence and presence of vorinostat (5 µM) for 6, 18 and 30 h, following starvation, with medium supplemented with 1% fetal bovine serum. (A) Notch receptor expression levels in OVCAR3 cells. RT-qPCR indicated that Notch2, Notch3 and Notch4 mRNA levels were increased following vorinostat exposure. (B) Representative staining of Notch receptors by immunofluorescence. Vorinostat slightly increased the protein expression of Notch receptors 2 and 4, and decreased Notch1 expression following exposure to vorinostat (magnification, ×200). Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue) and Notch receptors with fluorescein isothiocyanate (green). (C) Notch ligand expression levels in OVCAR3 cell line. RT-qPCR revealed that Dll1, Jagged1 and Jagged2 mRNA expression levels in cells treated with vorinostat were increased compared with cells not treated with vorinostat (magnification, ×200). Dll3 and Dll4 mRNA expression levels were not differentially expressed. (D) Notch downstream target gene expression levels. RT-qPCR revealed that Hes1, Hes5, Hey1 and Hey2 mRNA expression levels in cells treated with vorinostat were increased compared with cells without vorinostat treatment. Hes6 mRNA expression levels were not affected by vorinostat treatment. RT-qPCR was normalized to hypoxanthine phosphoribosyltransferase. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05; **P<0.01; ***P≤0.001 vs. control cells. Dll, Delta-like; RT-qPCR, reverse transpiration-quantitative polymerase chain reaction; Hes, hairy enhancer of split; Hey, Hes-related proteins.

Article Snippet: The primary antibodies were as follows: Rabbit polyclonal anti-human Notch1 extracellular (catalog no., ABS90; EMD Millipore, Billerica, MA, USA), rabbit monoclonal anti-Notch1 cleaved (catalog no., 4147; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-Notch2 cleaved (catalog no., 07-1234; EMD Millipore) and rabbit polyclonal anti-Notch4 (catalog no., N5163; Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Targeted Gene Expression, Standard Deviation, Control, Real-time Polymerase Chain Reaction

Journal: International Journal of Clinical and Experimental Pathology

Article Title: The expression of the nicotinic acetylcholine receptor α3 subunit in the brains of patients with Alzheimer’s disease and its effects on α- and γ-secretases and Notch signal transduction in SH-SY5Y cells

doi:

Figure Lengend Snippet: Hes and Notch1 levels in SH-SY5Y cells administered α3siRNA or nicotine. Hes mRNA (A) and protein (B) amounts in SH-SY5Y cells administered α3 siRNA or nicotine, as determined by real time PCR and Western blotting, respectively. Notch1 mRNA (C) and protein (D) amounts in SH-SY5Y cells administered α3 siRNA or nicotine, as assessed by Real time PCR and Western blotting, respectively. The data are presented as the mean ± SD (n = 9). **P < 0.01 vs control group.

Article Snippet: Goat polyclonal antibodies against α3nAChR (SC1771), rabbit polyclonal antibodies against α3nAChR (GTX105495), mouse polyclonal antibodies against Aβ (Biolegend 803001), horseradish peroxidase conjugated anti-mouse and anti-goat secondary antibodies, fluorescence (FITC)-labeled sheep anti-rabbit IgG, fluorescence (Cy3) labeled sheep against mouse IgG, mouse monoclonal antibodies against NCT (SC136003), ADAM10 (SC28358), PS-1 (SC365495), Notch1 (SC32745), and Hes1 (SC166378) (Santa Cruz Biotechnology Inc., USA) were used.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions

doi: 10.1210/clinem/dgaa096

Figure Lengend Snippet: E2A, HEB, and NOTCH1 expression in normal endometrium (control) and in paired eutopic (Eosis-Eu) and ectopic (Eosis-Ec) endometrium of women and baboons with endometriosis. (A, B) qRT-PCR analysis of E2A, HEB, and NOTCH1 mRNA expression levels in paired eutopic and ectopic endometrium of women (n = 9) and baboons (n = 5). (C, D) Immunohistochemical localization of E2A, HEB, and NOTCH1 on sections of normal endometrium and paired eutopic and ectopic endometrium from women (control, n = 5; Eosis, n = 9) and baboons (n = 5) with endometriosis. Brown shows positive staining. *P < 0.05; **P < 0.01. Scale bars, 25 µm.

Article Snippet: Rabbit anti-human polyclonal antibody against NOTCH1 (sc6014R) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions

doi: 10.1210/clinem/dgaa096

Figure Lengend Snippet: Recombinant IL-6-induced p38-MAPK activation mediates elevated E2A, HEB, and NOTCH1 expression. (A) Quantitative reverse transcriptase (qRT)-PCR analyses if the expression of E2A, HEB, and NOTCH1 mRNA in 12Z cells in the presence of recombinant IL-6 (10 ng/mL) for 0, 6, 12, and 24 hours. (B) Western blot analysis of E2A, HEB, and NOTCH1 in 12Z cells in the presence of recombinant IL-6 (10 ng/mL) for 0, 6, 12, and 24 hours. (C) qRT-PCR analyses of the expression of E2A, HEB, and NOTCH1 mRNA in 12Z cells treated with either vehicle (0.1% BSA in PBS) or recombinant IL-6 (10 ng/mL) for 24 hours in the absence (only DMSO) or presence of SB203580 (10 μM dissolved in DMSO). (D) Western blot analysis of p-p38 MAPK, p38 MAPK, E2A, HEB, and NOTCH1 expression in 12Z cells treated with either vehicle alone or recombinant IL-6 for 24 hours in the absence or presence of SB203580. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit anti-human polyclonal antibody against NOTCH1 (sc6014R) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Recombinant, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions

doi: 10.1210/clinem/dgaa096

Figure Lengend Snippet: NOTCH1 expression in 12Z cells following E2A and HEB inhibition. (A) mRNA levels of E2A, HEB, and NOTCH1 following E2A, HEB, or combined E2A and HEB inhibition in 12Z cells, in the absence or presence of IL-6 (10 ng/mL) for 24 hours (n = 3). (B) Protein levels of E2A, HEB, and NOTCH1 following E2A inhibition, HEB inhibition or combined E2A and HEB inhibition in 12Z cells, in the absence or presence of IL-6 (10 ng/mL) for 24 hours. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit anti-human polyclonal antibody against NOTCH1 (sc6014R) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Inhibition

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions

doi: 10.1210/clinem/dgaa096

Figure Lengend Snippet: Binding efficiency of E2A and HEB binding sites on the human NOTCH1 promoter is enhanced following IL-6 stimulation. (A, B) Predicted E2A and HEB binding site on the human NOTCH1 promoter. (C, D) Binding efficiency of E2A and HEB on the NOTCH1 promoter was detected by chromatin immunoprecipitation-quantitative PCR (n = 3). *P < 0.05.

Article Snippet: Rabbit anti-human polyclonal antibody against NOTCH1 (sc6014R) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions

doi: 10.1210/clinem/dgaa096

Figure Lengend Snippet: IL-6 promotes endometriotic lesions development in the mouse model of endometriosis by inducing expression of E2A, HEB, and NOTCH1 in the epithelial cells of endometriotic lesions. (A) Experimental schematic of induction of endometriosis and IL-6 treatment in the mouse model. (B) The lesions in a control mouse and a mouse treated with IL-6 (5 µg/injection twice pera week for 2 weeks). (C) The number of endometriotic lesions of IL-6 group was significantly higher than the vehicle group. (D) Immunohistochemical analysis of E2A, HEB, and NOTCH1 was performed on sections of endometriotic lesions from the mouse model of endometriosis (n = 4/group). Brown shows positive staining. *P < 0.05. Scale bars, 25 µm.

Article Snippet: Rabbit anti-human polyclonal antibody against NOTCH1 (sc6014R) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Injection, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions

doi: 10.1210/clinem/dgaa096

Figure Lengend Snippet: Working model. In the epithelium of endometriotic lesions, IL-6 stimulates p38MAPK phosphorylation to stabilize E-proteins (E2A, HEB) transcripts; E-proteins then bind to the NOTCH1 promoter to induce the expression of NOTCH1.

Article Snippet: Rabbit anti-human polyclonal antibody against NOTCH1 (sc6014R) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing