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: 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: Stem Cells (Dayton, Ohio)

Article Title: Thyroid Hormone‐Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis

doi: 10.1002/stem.2544

Figure Lengend Snippet: Expression profiles of genes involved in Notch signaling in X. laevis intestine during natural and T3‐induced metamorphosis. Quantitative real‐time RT‐PCR was performed using total RNAs prepared from the intestine of animals at indicated developmental stages ( A, C, E, G, I, K, M) or stage‐54 tadpoles after 10 nM T3 treatment ( B, D, F, H, J, L, N ). Levels of Hairy1 ( A, B ), Hairy2b ( C, D ), DLL1 ( E, F ), DLL3 ( G, H ), Jag1 ( I, J ), Jag2 ( K, L ), and Notch1 ( M, N ) mRNAs are shown relative to those of ribosomal protein L8 (rpL8) mRNA, with the values at stage 54 or 0‐day treatment set to 1. Error bars represent the SEM ( n = 7 for A–D ; n = 3 for E–H , J, M, N ; n = 8 for I, L ; n = 5 for K ). The values were analyzed by ANOVA followed by Scheffe's post hoc test whose results are shown only for the adjacent stages or days except for Notch1 ( M ). Asterisks indicate that the mRNA levels are significantly different. *, p < .05, **, p < .01; ns: not significant.

Article Snippet: The sections of the intestine from stage‐62 tadpoles were double‐immunostained with a mixture of the mouse anti‐human cytokeratin 19 (CK19) (1:100; Novocastra), which is a predominant cytokeratin in the adult Ep including the stem cells , and the rabbit anti‐human DLL1 (1:100; Abcam, Tokyo, Japan), the rabbit anti‐human Jag1 (1:50; Santa Cruz Biotechnology, Dallas, TX), or the rabbit anti‐human Notch1 (1:500; Rockland Inc., Gilbertsville, PA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Stem Cells (Dayton, Ohio)

Article Title: Thyroid Hormone‐Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis

doi: 10.1002/stem.2544

Figure Lengend Snippet: Correlation of the expression patterns of Notch pathway components with that of a stem/progenitor marker in the metamorphosing intestine. Cross‐sections of the intestine from tadpoles at stages 60 ( A, D ) and 61 ( B, E, G, I ), and stage‐54 tadpoles treated with 10 nM T3 for 3 ( C, F ) and 5 days ( H, J ) were hybridized with antisense Hairy1 ( A‐C ) or Hairy2b ( G, H ) probes. The corresponding serial sections were hybridized with antisense LGR5 probe ( D–F, I, J ) for comparison. Hairy1 is expressed in the adult epithelial stem (AE)/progenitor cells (A, arrowhead) when they first appear as the small islets expressing LGR5 ( D , arrow) at stage 60. These islets grew in size as metamorphosis proceeded and continued to co‐express Hairy1 ( B , arrowheads) and LGR5 ( E , arrows). During T3‐induced metamorphosis, Hairy1 ( C , arrowhead) is also expressed in the AE/progenitor cells expressing LGR5 ( F , arrow) after 3 days of T3 treatment. Some, although not all, cells expressing Hairy2b were in the connective tissue (CT) ( G , arrowheads) underlying the AE/progenitor cells expressing LGR5 ( I , arrows). Hairy2b is also expressed in the CT after 5 days of T3 treatment ( H , arrowheads), again, with some underlying the AE/progenitor cells ( J , arrows). Cross‐sections of the intestine from tadpoles at stage 62 were double‐immunostained with anti‐CK19 to detect the islet of AE/progenitor cells ( K–M , red) and anti‐DLL1 ( K , green), anti‐Jag1 ( L , green), or anti‐Notch1 ( M , green) followed by counterstaining with DAPI ( K–M , blue). DLL1 ( K , arrowheads) and Notch1 ( M , arrowheads) are expressed in the islets (arrows). The cells expressing Jag1 are scattered in both the larval Ep and CT with some just beneath the islet, but not in the islets ( L , arrowheads). The dashed‐lines indicate the boundary of the Ep and the CT. Scale bars = 20 μm. Abbreviations: AE, adult epithelial stem/progenitor cells, CT, connective tissue, Ep, epithelium.

Article Snippet: The sections of the intestine from stage‐62 tadpoles were double‐immunostained with a mixture of the mouse anti‐human cytokeratin 19 (CK19) (1:100; Novocastra), which is a predominant cytokeratin in the adult Ep including the stem cells , and the rabbit anti‐human DLL1 (1:100; Abcam, Tokyo, Japan), the rabbit anti‐human Jag1 (1:50; Santa Cruz Biotechnology, Dallas, TX), or the rabbit anti‐human Notch1 (1:500; Rockland Inc., Gilbertsville, PA).

Techniques: Expressing, Marker, Comparison

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