Journal:

Article Title: Ethanol Stimulates Endothelial Cell Angiogenic Activity via a Notch-Angiopoietin 1 dependent pathway

doi: 10.1093/cvr/cvn108

Figure Lengend Snippet: Endothelial cells were treated without (control) or with EtOH (25mM) for 24h. (a) QRTPCR analysis of Notch 1 and 4 receptors and Notch target gene hrt-1, 2 and 3. Data were normalized to GAPDH and represent the mean ± SEM values from three independent experiments. (b) Representative Western blots of Notch 1 and 4 IC showing increase in protein expression following ethanol treatment. (c) The effect of ethanol on CBF-1/RBP-Jk promoter activity determined by luciferase assay as described in Methods. Data represent the mean ± SEM, n=3. *P<0.05 vs control.

Article Snippet: Briefly, 2×10 5 cells were transfected with 2 μg of siRNA targeting Notch 1 or Notch 4 or Ang1 or Tie2 (Ambion, Austin, TX) or a scrambled negative control siRNA (Ambion, cat #4611) in 75 μl of siRNA electroporation buffer.

Techniques: Western Blot, Expressing, Activity Assay, Luciferase

Journal:

Article Title: Ethanol Stimulates Endothelial Cell Angiogenic Activity via a Notch-Angiopoietin 1 dependent pathway

doi: 10.1093/cvr/cvn108

Figure Lengend Snippet: HUVEC transfected with scrambled RNA (scrambled control) or with an siRNA targeted to Notch 1 or Notch 4 were treated without or with EtOH (25mM, 24h) before (a) Network formation on matrigel was assessed (cumulative data from 3 separate experiments conducted in triplicate shown. *P<0.05 vs scrambled control. #p<0.05 vs EtOH treated scrambled control) or (b) Ang1 and Tie2 mRNA levels were analyzed by QRTPCR. Data were normalized to GAPDH and represent the mean ± SEM values from three independent experiments. *P<0.05 vs scrambled control. #p<0.05 vs EtOH treated scrambled control.

Article Snippet: Briefly, 2×10 5 cells were transfected with 2 μg of siRNA targeting Notch 1 or Notch 4 or Ang1 or Tie2 (Ambion, Austin, TX) or a scrambled negative control siRNA (Ambion, cat #4611) in 75 μl of siRNA electroporation buffer.

Techniques: Transfection

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Sequences for primers used for reverse transcription-quantitative polymerase chain reaction analysis.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Notch1 and cleaved-Notch1 expression in the human prostatic carcinoma cell lines LNCaP, PC-3 and DU 145 and the immortalized human prostatic epithelial RWPE-1 cell line. (A) Western blot analysis. Total cell lysates were collected from LNCaP, PC-3, DU 145 and RWPE-1 cells, and analyzed for Notch1 and cleaved-Notch1 proteins. Anti-GAPDH antibody was included as a loading control. (B) Quantitative analysis for the changes of Notch1 and cleaved-Notch1 proteins. Intensity of the targeted protein/modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. *P<0.05 vs. RWPE-1 group.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control, Modification

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Knockdown of Notch1 by shRNA in LNCaP cells. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis of Notch1 mRNA and protein expression levels following transfection. (C) Quantitative analysis of Notch1 and cleaved-Notch1 protein levels. Intensity of the targeted protein and modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. *P<0.05 vs. control group. shRNA, short hairpin RNA.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Modification, Control

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Effect of Notch1-knockdown on invasion and proliferation of LNCaP cells. (A) Results of the Matrigel invasion assay and (B) quantitative analysis. *P<0.05 vs. control group. (C) Results of the cell proliferation assay, detected by WST-1 agent. Data represent the average results from three independent experiments. shRNA, short hairpin RNA.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Invasion Assay, Control, Proliferation Assay, shRNA

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Effect of Notch1-knockdown on the expression of genes involved in cell invasion in LNCaP cells, assessed by reverse transcription-quantitative polymerase chain reaction. Data represent the average results from three independent experiments. *P<0.05 vs. control group. MTA1, metastasis-associated 1; KISS-1, KiSS-1 metastasis-suppressor, MKK4, mitogen-activated protein kinase 4; KAI1, cluster of differentiation 82; shRNA, short hairpin RNA.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, shRNA

Journal: Cancers

Article Title: Long Non-Coding RNA-Based Functional Prediction Reveals Novel Targets in Notch-Upregulated Ovarian Cancer

doi: 10.3390/cancers14061557

Figure Lengend Snippet: Discovery of Notch-related lncRNAs in HGSC. ( A ) Results of GSEA using Hallmark gene sets in the TCGA HGS-OVCA database (N = 303) to validate our grouping according to NOTCH1/3 expression. “High” indicates a group with high expressions of both NOTCH1 and NOTCH3 ( n = 97). “Low” indicates a group with low expressions of both NOTCH1 and NOTHC3 ( n = 96). The classification into high and low was based on median TPM values. High NOTCH1 with low NOTCH3 ( n = 55) and high NOTCH3 with low NOTCH1 ( n = 55) were excluded from this analysis. Red coloured dots indicated statistically significant p -value (<0.05) and FDR q-value (<0.025). ( B ) Identification of differentially expressed lncRNAs using TANRIC database ( n = 293). Ten of the TCGA HGS-OVCA cases ( n = 303) did not show lncRNA expression. Heatmap showing representative lncRNAs ( n = 1757) according to NOTCH1/3 expression status. Based on the expression status of NOTCH1/3, TCGA HGS-OVCA dataset was divided into four groups: I ( n = 92), low expression of both NOTCH1 and 3; II ( n = 54), high expression of NOTCH3 and low expression of NOTCH1; III ( n = 54), high expression of NOTCH1 and low expression of NOTCH3; and IV ( n = 93), high expression of both NOTCH1 and 3. ( C ) Potential function of Notch-related lncRNAs using FuncPred. Red coloured dots indicate unique gene sets highly ranked in this analysis but not in GSEA depicted in ( A ). ( D ) Venn diagram presenting the logical relation between FuncPred data with lncRNAs and GSEA data with mRNAs. Abbreviations: NOTCH1/3, Notch receptor 1 and 3; TCGA, The Cancer Genome Atlas; HGS-OVCA, high-grade serous ovarian cancer; lncRNA, long non-coding RNA; GSEA, gene set enrichment analysis; FuncPred, in silico prediction of gene function for protein-coding and lncRNA human genes.

Article Snippet: Three different small interfering RNAs (siRNAs) targeting NOTCH1 (cat#4851-1, 4851-2, and 4851-3) and NOTCH3 (cat#4854-1, 4854-2, and 4854-3) were purchased from Bioneer (Deajeon, Korea).

Techniques: Expressing, In Silico

Journal: Cancers

Article Title: Long Non-Coding RNA-Based Functional Prediction Reveals Novel Targets in Notch-Upregulated Ovarian Cancer

doi: 10.3390/cancers14061557

Figure Lengend Snippet: Discovery of Notch-related lncRNAs using transcriptome data from OVCAR3 cells treated with siNOTCH1/3. ( A ) Heatmap showing differentially expressed lncRNAs ( n = 550) according to siNOTCH1/3 treatment. ( B ) Predicted function of Notch-related lncRNAs based on our siRNA experiments using FuncPred. Red coloured dots indicated unique gene sets highly ranked in this analysis, but not in GSEA, as depicted in A. ( C ) Venn diagram presenting the logical relation between upregulated lncRNAs in high NOTCH1/3 group in TCGA OVCA and downregulated lncRNAs in our siRNA experiments. ( D ) List of common lncRNAs, as depicted in ( C ). ( E ) Potential function of the common lncRNAs listed in ( D ). Red coloured dots indicate unique gene sets highly ranked in this analysis, but not in GSEA, as depicted in A. Abbreviations: TANRIC, The Atlas of Noncoding RNAs in Cancer.

Article Snippet: Three different small interfering RNAs (siRNAs) targeting NOTCH1 (cat#4851-1, 4851-2, and 4851-3) and NOTCH3 (cat#4854-1, 4854-2, and 4854-3) were purchased from Bioneer (Deajeon, Korea).

Techniques:

Journal: Cancers

Article Title: Long Non-Coding RNA-Based Functional Prediction Reveals Novel Targets in Notch-Upregulated Ovarian Cancer

doi: 10.3390/cancers14061557

Figure Lengend Snippet: Validation of in silico analysis and transcriptome data in HGSC cell line. Results of qRT-PCR for mRNAs of representative Notch-related lncRNAs ( A ), DNA repair-related targets ( B ), and Spermatogenesis-related targets ( C ) in OVCAR3 cells transfected with control siRNA (siControl) or NOTCH 1/3 siRNAs as indicated for 48 h. Comparisons of the relative expression of representative Notch-related lncRNAs ( D ), genes related to DNA repair ( E ), and spermatogenesis ( F ) in OVCAR3 cells following treatment with DAPT for 48 h. Data is shown as mean ± SD and p -values were calculated by two-tailed Mann–Whitney U-test. All experiments were repeated three times, and each experiment was performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: CTL, Control; TAF1C, TATA-Box Binding Protein Associated Factor; ADCY6, Adenylate Cyclase Type 6; DGCR8, DiGeorge Syndrome Critical Region Gene 8; RAD52, DNA Repair Protein RAD52 Homolog; ACRV1, Acrosomal Vesicle Protein 1; NPY5R, Neuropeptide Y Receptor Y5; DMC1, DNA Meiotic Recombinase 1; TCP11, T-Complex 11, DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester.

Article Snippet: Three different small interfering RNAs (siRNAs) targeting NOTCH1 (cat#4851-1, 4851-2, and 4851-3) and NOTCH3 (cat#4854-1, 4854-2, and 4854-3) were purchased from Bioneer (Deajeon, Korea).

Techniques: Biomarker Discovery, In Silico, Quantitative RT-PCR, Transfection, Control, Expressing, Two Tailed Test, MANN-WHITNEY, Binding Assay

Journal: Cancers

Article Title: Long Non-Coding RNA-Based Functional Prediction Reveals Novel Targets in Notch-Upregulated Ovarian Cancer

doi: 10.3390/cancers14061557

Figure Lengend Snippet: Validation of in silico analysis and transcriptome data in human ovarian tissue samples. ( A – C ) Comparisons of the relative expression of NOTCH1/NOTCH3 ( A ), representative Notch-related lncRNAs ( B ), genes related to DNA repair (TAF1C and ADCY6) and spermatogenesis ( C ). (ACRV1) using our own HGSC tissue samples according to NOTCH1/3 expression (high vs. low, n = 21 vs. n = 19, respectively). ( D – G ). Relative expression values of NOTC1/3 ( D ), notch-related lncRNAs ( E ), and representative genes related to DNA repair ( F ), and spermatogenesis ( G ), in benign or borderline ovarian tumors ( n = 16), clear cell ovarian carcinomas ( n = 5), and high-grade serous ovarian cancers (HGSC, n = 79). Data was shown as mean ± SD and p -values were calculated by two-tailed Mann–Whitney U-test. All experiments were repeated three times, and each experiment was performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: TAF1C, TATA-Box Binding Protein Associated Factor; ADCY6, Adenylate Cyclase Type 6; DGCR8, DiGeorge Syndrome Critical Region Gene 8; RAD52, DNA Repair Protein RAD52 Homolog; ACRV1, Acrosomal Vesicle Protein 1; NPY5R, Neuropeptide Y Receptor Y5; DMC1, DNA Meiotic Recombinase 1; TCP11, T-Complex 11.

Article Snippet: Three different small interfering RNAs (siRNAs) targeting NOTCH1 (cat#4851-1, 4851-2, and 4851-3) and NOTCH3 (cat#4854-1, 4854-2, and 4854-3) were purchased from Bioneer (Deajeon, Korea).

Techniques: Biomarker Discovery, In Silico, Expressing, Two Tailed Test, MANN-WHITNEY, Binding Assay

Journal: Cancers

Article Title: Long Non-Coding RNA-Based Functional Prediction Reveals Novel Targets in Notch-Upregulated Ovarian Cancer

doi: 10.3390/cancers14061557

Figure Lengend Snippet: Identification of common transcription factors for Notch-related lncRNAs and genes related to DNA repair and spermatogenesis. ( A ) Correlation analysis of common lncRNA clusters with genes related to DNA repair and spermatogenesis using data from TCGA OVCA and ( B ) our siRNA experiments. ( C ) Representative result of ENCODE predicting putative transcription factors. ( D ) Results from ENCODE using 21 out of 43 common lncRNAs presenting number of lncRNAs associated with putative transcription factors. ( E ) Results from ENCODE using genes related to DNA repair and spermatogenesis presenting number of genes associated with putative transcription factors. ( F ) Transcription factors that are up- or down-regulated according to NOTCH1/3 status. GSEA was performed using legacy subset of transcription target gene sets as described in the Methods section. Upper and lower panels presented the results of GSEA using TCGA OVCA and our transcriptome data, respectively. The legacy subset of transcription target gene sets originally supported 610 gene sets, but we excluded unknown transcription factor and applied gene set size filter (min = 15, max = 500), and finally used 466 gene sets. Each dot represents one transcription target gene set which showed adjusted p -value < 0.05 and FDR q-value < 0.25. The dotted line indicates FDR = 0.25. Abbreviations: EGR1, Early Growth Response 1; CCTF, CCCTC-Binding Factor; GABPA, GA Binding Protein Transcription Factor Subunit Alpha; E2F4, E2F Transcription Factor 4; FDR, False discovery rate.

Article Snippet: Three different small interfering RNAs (siRNAs) targeting NOTCH1 (cat#4851-1, 4851-2, and 4851-3) and NOTCH3 (cat#4854-1, 4854-2, and 4854-3) were purchased from Bioneer (Deajeon, Korea).

Techniques: Binding Assay

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Activated NOTCH1 is detected in valve cells in a flow dependent manner. (A) Normal human aortic valve section stained with an antibody specific to the active form of NOTCH1 (NICD, red). NICD was found in both endothelial (arrows) and interstitial cells (arrowheads). Autofluorescence (AutoFI, green) of collagen and elastin highlight the fibrosa layer (F) and ventricularis layer (V), respectively. Nuclei, DAPI (blue). Scale bars indicate 100 μm. (B) Schematic of experimental procedure. Normal HAVECs were transfected with control or NOTCH1 siRNA, then cultured in static or fluid flow conditions. Gene expression was compared by qRT-PCR or mRNA-seq. (C) qRT-PCR analysis of HAVECs from four conditions. NOTCH1, two canonical direct targets, HES1 and HEY2 and a known flow responsive gene, KLF2, were analyzed. Graphs show mean gene expression relative to the static, control siRNA condition with error bars representing standard deviation. (n=3; *, p<0.05; ** p<0.01; *** p<0.001; NS, Not Significant).

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Staining, Transfection, Cell Culture, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Pathways Activated by Shear Stress in NOTCH1 Dependent Manner

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Activation Assay, Coagulation

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Expression of endochondral ossification genes is affected by shear stress and NOTCH1 signaling. Graphs show mean gene expression relative to the static, control siRNA condition with error bars representing standard deviation. All numbers are shown in log2 scale. (n=3; *, p< 0.05; **, p< 0.01; ***, p< 0.001, NS, Not Significant).

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Expressing, Standard Deviation

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Heatmap and clustering analysis of RNA-seq and ChIP-seq from HAVECs reveal likely NOTCH1 direct targets. (A) The expression of each gene was normalized to the static, control siRNA condition and then Log2 transformed. Displayed genes were selected as matching one of two patterns reflecting coordinate regulation by both NOTCH1 and shear stress: Pattern 1) at least 2-fold down upon NOTCH1 siRNA knockdown in both static and flow conditions, and at least 2-fold up in flow control siRNA condition compared to static, or Pattern 2) at least 2 fold up in NOTCH1 siRNA knockdown in both static and flow conditions, and at least 2 fold down in flow control siRNA condition compared to static. (B) ChIP-seq score shows the value for the highest ChIP peak +/− 20 kb from the transcriptional start site (TSS) of each gene. Annotated genes represent potential direct targets of NOTCH1 based on ChIP-seq score.

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Expressing, Transformation Assay

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: MGP expression is NOTCH1 dependent. (A) Normal human aortic valve sections stained with antibodies specific to: i. cMGP, the active, carboxylated form of MGP; ii. ucMGP, the inactive, uncarboxylated form of MGP; and iii. phosphoSMAD 1/5/8 (pSMAD1/5/8) cMGP, ucMGP and pSMAD1/5/8 (red) were found in both endothelial (arrows) and interstitial (arrowheads) cells. Autofluorescence (AutoFI, green) of collagen and elastin highlight the fibrosa layer (F) and ventricularis layer (V), respectively. Nuclei, DAPI (blue). Scale bars indicate 100 μm. (B) qRT-PCR analysis of MGP mRNA expression in HAVECs under four conditions. Graph shows mean gene expression relative to the no flow, no siRNA condition with error bars representing the standard deviation. (n=3; *, p< 0.05). (C) Western blot detecting NICD, active MGP (cMGP) and GAPDH from HAECs infected in vitro with increasing amounts of myc-tagged NOTCH1 intracellular domain (NICD). (D) Immunostaining for NICD (red) in cultured Human Aortic Endothelial Cells (HAEC) or Human Aortic Interstitial Cells (HAIC). (E) qRT-PCR to measure MGP mRNA in control or NOTCHI-overexpressing HAICs. (F) Western blot detecting active MGP in control or γ-secretase inhibitor (DAPT) treated HAECs. (G) Quantification of cMGP protein in (F) normalized to Actin. Error bars show standard deviation (n=3; **, p< 0.01; ***, p< 0.001; NS, Not Significant).

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Expressing, Staining, Quantitative RT-PCR, Standard Deviation, Western Blot, Infection, In Vitro, Immunostaining, Cell Culture

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: NOTCH directly regulates MGP through an endothelial enhancer. (A) NICD1-myc ChIP-qPCR. Relative enrichment is shown for four sites: validated HES1 NOTCH1/CSL binding site, validated HES1 non-CSL binding site, predicted CSL binding sites within the 821 bp putative MGP enhancer and an MGP non-CSL site. The HES1 and MGP non-CSL binding sites are located approximately 2 kb from the CSL binding sites. Error bars indicate standard deviation. (n=4; *, p< 0.05). (B): EMSA snowing CSL binding to putative MGP enhancer. A 46 bp oligo corresponding to the NICD1-myc ChIP-seq peak was labeled with 32P-dCTP (MGP Probe). Incubation with in vitro transcribed and translated CSL (CSL TnT) shifted the probe (arrow) This interaction could be competed by addition of unlabeled MGP probe (Unlabeled MGP) or oligo corresponding to a validated HES1 enhancer containing CSL sites (Unlabeled HES1), but not with unlabeled oligos in which the CSL sites were mutated (Mut), demonstrating specificity. Reticulocyte lysate (Empty TnT) caused a higher, non-specific shift. (C) Images of whole mount or histological sections from E16.0 transgenic embryos containing the 821 bp MGP enhancer upstream of the Hsp68 minimal promoter driving LacZ with or without mutation of the three predicted CSL binding sites. Mice containing the wildtype (Wt) enhancer had strong endothelial cell expression of LacZ (blue) in the large vessels of the arterial system (8/13) and some weak expression in the aortic valve (arrows) (4/13 embryos). Mutation of CSL sites (Mut) abolished endothelial expression (8/8); the mutant enhancer had weak expression in the smooth muscle layer (arrowhead) of 3/8 embryos. Sections were counterstained with Eosin (red). Aorta, Ao; branchiocephalic artery, BC; left common carotid, LCC; aortic valve, AoV.

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Binding Assay, Standard Deviation, ChIP-sequencing, Labeling, Incubation, In Vitro, Transgenic Assay, Mutagenesis, Expressing

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Model of NOTCH1 regulation of human endothelial cell calcification. Yellow shapes indicate different classes of molecules and their sub-cellular localization. Genes in black are regulated by shear stress and NOTCH1 signaling, while those in orange also have a significant NOTCH1 ChIP-seq peak within 20 kb of the transcriptional start site, suggesting potential direct transcriptional regulation by NOTCH1.

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: ChIP-sequencing

Journal: BMC Genomics

Article Title: Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties

doi: 10.1186/s12864-023-09659-w

Figure Lengend Snippet: Disruption of glioblastoma stem-like properties by siRNA knockdown of genes with H4K5acK8ac-preferred promoters. A and B Comparative ChIP-seq occupancy tracks of H3K4me3, H4K5acK8ac, H3K27ac, and BRD4 at representative loci, in the presence or absence of JQ1. The promoter regions of ZNF883 ( A ) and RFX4 ( B ) were specifically enriched with H4K5acK8ac in 0316-GSC cells. The unique enrichment of H4K5acK8ac at promoters is highlighted in red. ChIP-seq reads were averaged from two biological replicates. C – E Disruption of gene expression by siRNA knockdown. C Efficiencies of siRNA knockdown of genes from Group 6 with H4K5acK8ac-preferred promoters and the GSC-specific control marker ( NOTCH1 ) are compared with the negative control ( si-NC ) in 0316-GSC cells ( n = 3). D Short-term proliferation assay of 0316-GSC cells subjected to siRNA knockdown. Cell proliferation rates at 7 days after siRNA knockdown of the selected genes are shown ( n = 6). E Expression of stem cell marker genes, NESTIN and SOX2 , following siRNA knockdown of the selected genes ( n = 3). F and G Sphere formation assay following siRNA knockdown of the selected genes. F Phase-contrast images of 0316-GSC cells treated with target-specific siRNA. Images are representative of three independent experiments. Scale bar, 50 μm. G In vitro sphere formation efficiency of 0316-GSC cells treated with siRNA for 2 weeks ( n = 3). C – E and G Data are means ± SEM ** P < 0.01 (two-tailed Student’s t -test)

Article Snippet: For siRNA-mediated knockdown of gene expression, 0316-GSC, U87, and C13NJ cells were transfected with 25 nM siRNA targeting a gene of interest, negative control siRNA (Cat. No. AM4611, ThermoFisher Scientific), or NOTCH1-siRNA (Applied Biosystems) by using Lipofectamine RNAiMAX Transfection Reagent (Cat. No. 13778030, ThermoFisher Scientific).

Techniques: Disruption, ChIP-sequencing, Expressing, Marker, Negative Control, Proliferation Assay, Tube Formation Assay, In Vitro, Two Tailed Test

Journal:

Article Title: Ethanol Stimulates Endothelial Cell Angiogenic Activity via a Notch-Angiopoietin 1 dependent pathway

doi: 10.1093/cvr/cvn108

Figure Lengend Snippet: HUVEC transfected with scrambled RNA (scrambled control) or with an siRNA targeted to Notch 1 or Notch 4 were treated without or with EtOH (25mM, 24h) before (a) Network formation on matrigel was assessed (cumulative data from 3 separate experiments conducted in triplicate shown. *P<0.05 vs scrambled control. #p<0.05 vs EtOH treated scrambled control) or (b) Ang1 and Tie2 mRNA levels were analyzed by QRTPCR. Data were normalized to GAPDH and represent the mean ± SEM values from three independent experiments. *P<0.05 vs scrambled control. #p<0.05 vs EtOH treated scrambled control.

Article Snippet: Briefly, 2×10 5 cells were transfected with 2 μg of siRNA targeting Notch 1 or Notch 4 or Ang1 or Tie2 (Ambion, Austin, TX) or a scrambled negative control siRNA (Ambion, cat #4611) in 75 μl of siRNA electroporation buffer.

Techniques: Transfection

Journal:

Article Title: Ethanol Stimulates Endothelial Cell Angiogenic Activity via a Notch-Angiopoietin 1 dependent pathway

doi: 10.1093/cvr/cvn108

Figure Lengend Snippet: HUVEC mock transfected (p7pcmv) or transfected with RPMS-1 (an Epstein Barr virus-encoded gene product that binds CBF-1) were treated with or without EtOH (25mM, 24 h) before their network formation on Matrigel and Ang1, Tie2 expression were determined. (a) Network formation data (cumulative data, n=3). (b) QRTPCR analysis of Ang1 and Tie2 mRNA. Data were normalized to GAPDH and represent the mean ± SEM values from three independent experiments. *P<0.05 vs mock transfected, #p<0.05 vs EtOH treated mock transfected. (c) siRNA-directed knockdown of Ang1 and Tie2 inhibits ethanol-stimulated network formation. Representative Western blots for Angiopoietin 1 (Ang1) and Tie2 protein following respective siRNA knockdown. HUVEC transfected with scrambled RNA (control) or with an siRNA targeted to Ang1 or Tie2 were treated with or without ethanol (EtOH, 25 mM, 24 h) before their network formation on Matrigel was determined. *P<0.05 vs control, #p<0.05 vs EtOH treated control.

Article Snippet: Briefly, 2×10 5 cells were transfected with 2 μg of siRNA targeting Notch 1 or Notch 4 or Ang1 or Tie2 (Ambion, Austin, TX) or a scrambled negative control siRNA (Ambion, cat #4611) in 75 μl of siRNA electroporation buffer.

Techniques: Transfection, Expressing, Western Blot

Journal: BMC Genomics

Article Title: Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties

doi: 10.1186/s12864-023-09659-w

Figure Lengend Snippet: Disruption of glioblastoma stem-like properties by siRNA knockdown of genes with H4K5acK8ac-preferred promoters. A and B Comparative ChIP-seq occupancy tracks of H3K4me3, H4K5acK8ac, H3K27ac, and BRD4 at representative loci, in the presence or absence of JQ1. The promoter regions of ZNF883 ( A ) and RFX4 ( B ) were specifically enriched with H4K5acK8ac in 0316-GSC cells. The unique enrichment of H4K5acK8ac at promoters is highlighted in red. ChIP-seq reads were averaged from two biological replicates. C – E Disruption of gene expression by siRNA knockdown. C Efficiencies of siRNA knockdown of genes from Group 6 with H4K5acK8ac-preferred promoters and the GSC-specific control marker ( NOTCH1 ) are compared with the negative control ( si-NC ) in 0316-GSC cells ( n = 3). D Short-term proliferation assay of 0316-GSC cells subjected to siRNA knockdown. Cell proliferation rates at 7 days after siRNA knockdown of the selected genes are shown ( n = 6). E Expression of stem cell marker genes, NESTIN and SOX2 , following siRNA knockdown of the selected genes ( n = 3). F and G Sphere formation assay following siRNA knockdown of the selected genes. F Phase-contrast images of 0316-GSC cells treated with target-specific siRNA. Images are representative of three independent experiments. Scale bar, 50 μm. G In vitro sphere formation efficiency of 0316-GSC cells treated with siRNA for 2 weeks ( n = 3). C – E and G Data are means ± SEM ** P < 0.01 (two-tailed Student’s t -test)

Article Snippet: For siRNA-mediated knockdown of gene expression, 0316-GSC, U87, and C13NJ cells were transfected with 25 nM siRNA targeting a gene of interest, negative control siRNA (Cat. No. AM4611, ThermoFisher Scientific), or NOTCH1-siRNA (Applied Biosystems) by using Lipofectamine RNAiMAX Transfection Reagent (Cat. No. 13778030, ThermoFisher Scientific).

Techniques: Disruption, ChIP-sequencing, Expressing, Marker, Negative Control, Proliferation Assay, Tube Formation Assay, In Vitro, Two Tailed Test

Journal: BMC Genomics

Article Title: Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties

doi: 10.1186/s12864-023-09659-w

Figure Lengend Snippet: Disruption of glioblastoma stem-like properties by siRNA knockdown of genes with H4K5acK8ac-preferred super-enhancers. A – C Disruption of genes by siRNA knockdown. A siRNA knockdown of MYCN and NFIC compared with the negative control ( si-NC ) in 0316-GSC cells ( n = 3). B Short-term proliferation assay of 0316-GSC cells subjected to siRNA knockdown. Cell proliferation rates at 7 days after siRNA knockdown of MYCN and NFIC are shown ( n = 6). C Expression of stem cell marker genes, NESTIN (left) and SOX2 (right), following siRNA knockdown of MYCN and NFIC ( n = 3). D Sphere formation assay. Phase-contrast images of 0316-GSC cells treated with si-MYCN , si-NFIC , or si-NC for 2 weeks. Images are representative of three independent experiments. Scale bar, 50 μm. E Quantitation of sphere formation results for 0316-GSC cells ( n = 3). A – C and E Data are means ± SEM ** P < 0.01 (two-tailed Student’s t -test)

Article Snippet: For siRNA-mediated knockdown of gene expression, 0316-GSC, U87, and C13NJ cells were transfected with 25 nM siRNA targeting a gene of interest, negative control siRNA (Cat. No. AM4611, ThermoFisher Scientific), or NOTCH1-siRNA (Applied Biosystems) by using Lipofectamine RNAiMAX Transfection Reagent (Cat. No. 13778030, ThermoFisher Scientific).

Techniques: Disruption, Negative Control, Proliferation Assay, Expressing, Marker, Tube Formation Assay, Quantitation Assay, Two Tailed Test