Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: A) The purity of isolated cells was tested using anti-Notch1 antibody (green). Propidium iodide (PI, red) was used to visualize the nucleus of the cell. Bar is 50 μm. B) The levels of Notch1 expression in isolated cells were higher compared to expression of Notch1 in whole retinas. Gene expression was measured by qRT-PCR. Results are expressed as a percentage of the corresponding value in the Notch1 + cells isolated from embryonic day 14 developing retinas ± SEM (*P < 0.05).

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Isolation, Expressing, Gene Expression, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: Functional annotation for the selected genes revealed by One-Way ANOVA.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Functional Assay

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: For each gene, results are expressed as percentages ± SEM of the corresponding values in the Notch1 + cells isolated from E14 developing retinas (*P < 0.05).

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Isolation

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: The expression of retinal cell markers in Notch1 + progenitor cells and in whole retina samples at E14 and P0.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: The putative members of E14 Notch1 and P0 Notch1 gene networks.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques:

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: List of RT-PCR primers.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques:

Journal: Nutrients

Article Title: Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

doi: 10.3390/nu8070439

Figure Lengend Snippet: Primers for reverse transcriptase polymerase chain reaction RT-PCR.

Article Snippet: The membranes were subsequently blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and were incubated with primary antibodies against cytokeratin 20 (CK20, Abcam, Cambridge, MA, USA), Notch1 (Novus Biologicals, Littletown, CO, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p-GSK3β (Cell Signaling Technology, Danvers, MA, USA) overnight.

Techniques: Reverse Transcription, Polymerase Chain Reaction

Journal: Nutrients

Article Title: Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

doi: 10.3390/nu8070439

Figure Lengend Snippet: WPE and its bioactive compounds suppress colon CSCs markers, including CD133, CD44, DLK1, and Notch1 as well as Wnt/β-catenin signaling in colon CSCs. CD133 + CD44 + HCT116 cells were treated with varying concentrations of WPE (0, 10, 20, and 40 μg/mL) ( A a , Ba ); or concentrations of (+)-catechin, chlorogenic acid, ellagic acid and gallic acid comparable to 40 μg/mL of WPE ( A b , Bb ) for 6 days. mRNA expressions of CD133, CD44, DLK1 and Notch1 were examined by RT-PCR, and β-actin was used as a loading control ( A ); Protein levels of Notch1, β-catenin and p-GSK3β were examined by Western blot analysis, and α-tubulin was used as a loading control ( B ). Representative blots are shown in left panel and quantified in right panel. The values shown are the means ± SEM. A p -value < 0.05 was considered significant. Ctrl, Control; WPE, walnut phenolic extract; CSCs, cancer stem cells; CAT, (+)-catechin; CGA, chlorogenic acid; EA, ellagic acid; GA, gallic acid.

Article Snippet: The membranes were subsequently blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and were incubated with primary antibodies against cytokeratin 20 (CK20, Abcam, Cambridge, MA, USA), Notch1 (Novus Biologicals, Littletown, CO, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p-GSK3β (Cell Signaling Technology, Danvers, MA, USA) overnight.

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

Journal: Nutrients

Article Title: Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

doi: 10.3390/nu8070439

Figure Lengend Snippet: WPE down-regulates CD133, CD44, DLK1, and Notch1 in human primary cells from colorectal cancer tissue. Primary cancer cells were treated with varying concentrations of WPE (0, 10, 20, and 40 μg/mL) for 3 d. mRNA expressions of CD133, CD44, DLK1 and Notch1 were detected by RT-PCR, and GAPDH was used as a loading control. The values shown are the means ± SEM. A p -value < 0.05 was considered significant. Ctrl, Control.

Article Snippet: The membranes were subsequently blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and were incubated with primary antibodies against cytokeratin 20 (CK20, Abcam, Cambridge, MA, USA), Notch1 (Novus Biologicals, Littletown, CO, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p-GSK3β (Cell Signaling Technology, Danvers, MA, USA) overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Notch was found up-regulated after azelaic acid (AZA) treatment. (A) 120 differential expressed proteins associated in 52 functions after AZA treatment by protein mass spectrometry analysis in the heat map. (B) GO annotation with 528 differential expressed proteins. (C, D) THP-1, Molm-13, NK, and T cells were treated with 10 µM AZA for 24 h, the expression level of ICN1 and ICN2 was validated by western blot. (E) The expression of Notch1 and Notch2 in mouse spleen measured by ICH. IHC, immunohistochemistry.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Mass Spectrometry, Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Azelaic acid (AZA) exerts anti-leukemic effect by activating the Notch signaling pathway. (A) Notch responsive elements were transfected into 293T cells after 24 h. Cells were then treated with 10 µM AZA, 10 µM RO4929097, and combination for 24 h, the Notch signaling reporter assay was measured by dual luciferase reporter activity. (B) Validation of the RNA expression of Notch1 and Notch2, the downstream target genes HES1 and HEY1 in Molm-13 and THP-1 cells by qPCR. (C) The protein expression level of ICN1, ICN2, HEY1, and HES1 in acute myeloid leukemia (AML) cell lines after treatment of AZA and RO4929097 and their detection by western blot. ImageJ was used for the densitometric analysis. Data represent means ± SD. (D) Molm-13 cells were pretreated with 10 µM RO4939097 for 24 h, and then treated with 10 µM AZA for another 24 h. Cells were collected for apoptosis analysis. (E) NK cells and T cells were pretreated with 10 µM AZA, 10 µM RO4929097, and combination for 24h before co-culture with THP-1 cell and Molm-13 cells at an E:T ratio of 5:1. The cytotoxicity of NK and T was determined by detecting the LDH release rate. (F) NK and T cells were pre-treated with AZA and RO4929097, then co-cultured with THP-1 cell at an E:T ratio of 3:1 for 4 h. The level of TNF-α and IFN-γ in the supernatant was measured by ELISA. A Total of three independent experiments were performed. *P < 0.05, **P < 0.01,***P < 0.001.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Transfection, Reporter Assay, Luciferase, Activity Assay, Biomarker Discovery, RNA Expression, Expressing, Western Blot, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed by thalidomide

doi: 10.1038/srep27280

Figure Lengend Snippet: ( A ) The expression of HIF-1α and HIF-2α in gastrointestinal vascular malformations and normal vessels. Red arrow: strongly positive; Black arrow: weakly positive; Blue arrow: negative. ( B ) Percentages of positive and negative vessels in GIVM and normal tissues. ** P < 0.01.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA), which was blocked using 5% milk for 2 h, and incubated with rabbit-anti-HIF-2α, VEGF, NOTCH1, DLL4, Ang2, and β-actin antibodies (all rabbit polyclonal antibodies from KangChen Bio-tech, Shanghai, China) at 4 °C overnight.

Techniques: Expressing

Journal: Scientific Reports

Article Title: HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed by thalidomide

doi: 10.1038/srep27280

Figure Lengend Snippet: ( A ) Western blot determinations of HIF-1α and HIF-2α expression at different time points of hypoxia. * P < 0.05, ** P < 0.01 vs. 0 hour. ( B ) The effect of HIF-1α and HIF-2α overexpression on the expression of VEGF, Notch1, DLL4, and Ang2. Western blot and RT-PCR demonstrated that HIF-1α and HIF-2α overexpression increased the expression of VEGF, Notch1, DLL4, and Ang2 protein and mRNA. * P < 0.05, ** P < 0.01 vs. control. ( C ) Influence of HIF-1α and HIF-2α overexpression on angiogenesis 6 and 24 h after transfection of Lenti-HIF-1α and Lenti-HIF2α. Tube formation was enhanced 6 and 24 h after transfection. ** P < 0.01 vs. control. ( D ) Fluorescence microscope observations of subintestinal vein sprouting in normal and HIF-2α-overexpressing zebrafish. *Indicates subintestinal vascular sprouts. HIF-2α overexpression significantly increased the number of subintestinal vascular sprouts. ** P < 0.01 vs. control plasmid. ( E ) Dual luciferase reporter gene assay demonstrated that HIF-2α enhanced VEGF promoter activity. ** P < 0.01 vs. control plasmid.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA), which was blocked using 5% milk for 2 h, and incubated with rabbit-anti-HIF-2α, VEGF, NOTCH1, DLL4, Ang2, and β-actin antibodies (all rabbit polyclonal antibodies from KangChen Bio-tech, Shanghai, China) at 4 °C overnight.

Techniques: Western Blot, Expressing, Over Expression, Reverse Transcription Polymerase Chain Reaction, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Luciferase, Reporter Gene Assay, Activity Assay

Journal: Scientific Reports

Article Title: HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed by thalidomide

doi: 10.1038/srep27280

Figure Lengend Snippet: ( A ) Immunofluorescence indicated that HIF-1α and HIF-2α expression was down-regulated by thalidomide at different concentrations. ( B ) Western blots demonstrated that the expression of HIF-1α and HIF-2α decreased with 100 and 200 μg/ml of thalidomide. * P < 0.05, ** P < 0.01. ( C ) Western blots demonstrated that thalidomide at 200 μg/ml inhibited the expression of HIF-1α and HIF-2α in HUVECs after hypoxic treatment for 24, 36, and 48 h. * P < 0.05, ** P < 0.01. ( D ) Fluorescence microscope observations of the effect of thalidomide at different concentrations on vascular development in zebrafish with HIF-2α overexpression. ** P < 0.01 vs. HIF-2α.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA), which was blocked using 5% milk for 2 h, and incubated with rabbit-anti-HIF-2α, VEGF, NOTCH1, DLL4, Ang2, and β-actin antibodies (all rabbit polyclonal antibodies from KangChen Bio-tech, Shanghai, China) at 4 °C overnight.

Techniques: Immunofluorescence, Expressing, Western Blot, Fluorescence, Microscopy, Over Expression

Journal: OncoTargets and therapy

Article Title: Anticancer activities of epigallocatechin-3-gallate against cholangiocarcinoma cells

doi: 10.2147/OTT.S112364

Figure Lengend Snippet: Antitumor activities of EGCG against HuCC-T1 tumor xenograft mice model. Notes: ( A ) Tumor growth. 1×10 7 cells were subcutaneously injected to the back of BALB/c nude mice. When the diameter of the solid tumor reached about 4 mm, EGCG in a vehicle (hydrogel) was injected subcutaneously beside the solid tumor (dose: 20 mg EGCG/kg). For comparison, the vehicle as a control was injected into the back of the mice. Growth of the tumor was calculated using the formula V = ( a × [ b ] )/2, with a being the largest and b being the smallest diameter. For immunohistochemistry of tumor tissues, tumors were isolated and fixed with 4% formamide 25 days after the injection. ( B ) Immunohistochemistry (400×) of tumor tissues. Notch 1, MMP-2 and -9, and PCNA antibodies were used for staining tumor tissues. * P <0.01. Abbreviations: EGCG, epigallocatechin-3-gallate; HuCC-T1, human cholangiocellular carcinoma cell line; MMP, matrix metalloproteinase; PCNA, proliferating cell nuclear antigen.

Article Snippet: For immunohistochemical staining, anti-Notch1 antibody (Enzo Life Sciences Inc, ADI-905-897, Farmingdale, NY, USA), anti-MMP-2 antibody (SC-10736; Santacruz Biotech.

Techniques: Injection, Immunohistochemistry, Isolation, Staining