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: 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 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: bioRxiv

Article Title: Notch engagement by Jag1 nanoscale clusters indicates a force-independent mode of activation

doi: 10.1101/2022.11.22.517517

Figure Lengend Snippet: (A) A rod like DNA origami was used to create 1x, 2x, 3x, 4x and 8x Jag1Fc nanopatterns (JNPs). (B) Gel retardation assay confirms an increasing molecular weight when increasing the number of proteins positioned on top of the DNA origami. 1Kb ladder (L), scaffold (Sc), 0x JNP (0), repeat of (0) but with added Jag1Fc-DNA conjugates (control for non-specific binding), structures loaded with Jag1Fc patterns: 1x, 2x, 3x, 4x and 8x JNP run on 2% agarose gel stained with ethidium bromide. (C) Schematic representation of the DNA origami used for the DNA PATNT experiments, in which the Jag1 proteins conjugated to DNA-handles also contain an extension for DNA-PATNT docking sites used for direct Jag1Fc-DNA conjugate detection (red). (D) Average (cyan, thick) and individual cropped DNA PATNT superresolution images (white, thin) of JNPs (scale bars = 50nm) with the bar graphs showing the Jag site occupancy distributions of the different nanorods. (E) Zoom-ins of negative stain TEM of (unpurified) 4x JNPs and empty DNA origami rods (0x JNP) and zoom-out of 4x JNPs (bottom). Scale bars are 100 nm. (F) Avidity effect of JNPs. Surface Plasmon Resonance measurements using Notch1 (EGF8-12) receptor immobilized on chip the surface and increased concentrations of 1x, 2x, 3x, 4x and 8x JNP flow through the chip. The mean apparent kD of different Jag1Fc nano-patterns is shown on a bar plot where dots represent two individual repeats.

Article Snippet: The rabbit TgG monoclonal antibody for the detection of cleaved Notch1 (Val1744) (D3B8) (Cell Signalling Technology, cat. no. 4147) was diluted 1:200 in 1x Duolink Antibody Diluent (Sigma Aldrich, cat. no. DUO92002).

Techniques: Electrophoretic Mobility Shift Assay, Molecular Weight, Control, Binding Assay, Agarose Gel Electrophoresis, Staining, SPR Assay

Journal: bioRxiv

Article Title: Notch engagement by Jag1 nanoscale clusters indicates a force-independent mode of activation

doi: 10.1101/2022.11.22.517517

Figure Lengend Snippet: (A) DNA nanopatterns (NP) containing 0x, 1x, 2x, 3x, 4x and 8x Jag1Fc (JNPs) were used to stimulate iPSc-derived neuroepithelial stem-like (lt-NES) cells. (B) iPS cells shown in i) brightfield and ii) fluorescence channel after immunostaining with antibody against the extracellular part of Notch1 (magenta), F-actin (green) and nucleus (blue). Scale bars, 50 μm (C) iPS cells stimulated with 3x JNP for 1, 2, 3, 4, 5, 6 and 7 hours. Relative expression of Hes1 performed with primers for HES1 and GAPDH genes. (D) Bar plot of 1x, 2x, 3x and 4x JNP induced activation of Notch pathway detected after RNA extraction and qPCR with primers against HES1 and GAPDH genes. Data points represent 4 biological repeats. One way analysis of variance (ANOVA) was followed by Dunnett multiple-comparison test (**P< 0.01, ****P<0.0001). (E) iPS cells stimulated with 1x, 2x, 3x, 4x and 8x JNP at 0.03, 0.16, 0.3, 1.66, 3.33 and 6.66 nM final concentrations. RNA extracted and qPCR assay performed with primers for HES1 and GAPDH genes. (F) Proximity Ligation Assay (PLA) performed using antibody against cleaved NTCD on iPS cells treated either with 0x JNP or 8x JNP. Representative images of cells for each condition show with dots the activation signal (magenta), F-actin (green) and nucleus (blue). Scale bars, 10 μm. (G) Violin plot of the PLA experiment when detection signal is measured for 50 cells for each condition. One way analysis of variance (ANOVA) was followed by Tukey multiple-comparison test (**P< 0.01). (H) Heat map diagram of mRNA sequencing experiment performed on iPS cells after stimulation with 0x, 1x and 8x JNP and three biological repeats for each condition shown for genes with FDR < 0.05 and absolute log2FC > 0.5Data is shown automatically clustered using hierarchical complete-linkage clustering of euclidean distances. (I) Depicted Notch pathway related genes and transcripts per million (TPM) plotted for each condition. (J) Volcano plotsof genes upregulated by 1x JNP (1) and 8x JNP (8) relative to 0x JNP (0).

Article Snippet: The rabbit TgG monoclonal antibody for the detection of cleaved Notch1 (Val1744) (D3B8) (Cell Signalling Technology, cat. no. 4147) was diluted 1:200 in 1x Duolink Antibody Diluent (Sigma Aldrich, cat. no. DUO92002).

Techniques: Derivative Assay, Fluorescence, Immunostaining, Expressing, Activation Assay, RNA Extraction, Comparison, Proximity Ligation Assay, Sequencing

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: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: (a) Cytotoxicity analysis of the lipopeptides_Notch1 complex (MR 50 : 1) at the 72 h time point in the MDA-MB-231 breast cancer cell line compared to the HiPerFect_siRNA complex. Error bars indicate SEM from three separate replicates. (b) Flow cytometry data of time-dependent (1 h) cellular uptake studies of FAM-labelled lipopeptides (1.25 μM) in MDA-MB-231 cells and (c) flow cytometry analysis of cellular uptake studies of labelled siRNA complexes at MR 50 after 1 h of incubation in MDA-MB-231 cells. Among the designed lipopeptides, lipopeptide AB18 demonstrates the highest siRNA internalization in MDA-MB-231 cells; error bars indicate SEM from two separate replicates (*** p < 0.0001, ** p < 0.01 and n.s. – not significant compared with untreated cells). (d) Intracellular distribution of the lipopeptide_siRNA complex (MR 50) in MDA-MB-231 cells after 6 h of incubation by ApoTome microscopy. Nuclei are stained with DAPI (blue), green represents the FAM-labelled lipopeptide, and red represents DY-547 labelled siRNA and also represents the released siRNA from lipopeptide_siRNA complexes. Yellow represents the co-localization of the FAM-labelled lipopeptide and DY-547-labelled siRNA. The best cytosolic distribution of siRNA was seen in the case of the lipopeptide AB18_siRNA complex. MDA-MB-231 cells incubated with HiPerFect_siRNA has less cytoplasmic distribution of siRNA and exhibits a less healthy morphology of MDA-MB-231 cells compared to the lipopeptide AB18_siRNA complex. (e) Intracellular distribution of the lipopeptide AB36_siRNA complex (MR 50) and HiPerFect_siRNA complex after 6 h of incubation in the hard to transfect primary cell line HUVEC by ApoTome microscopy. Please note that lipopeptide AB36 has a similar peptide sequence to lipopeptide AB18, but unlike lipopeptide AB18, it does not have FAM to enable microscopic studies without interference. Nuclei are stained with DAPI (blue), and red represents DY-547 labelled siRNA. (f) Flow cytometry analysis of cellular uptake studies of labelled siRNA complexes at MR 50 after 1 h of incubation in the hard to transfect primary cell line HUVEC. Error bars indicate SEM from two separate replicates. Both (e) and (f) indicate that labelled siRNA is internalized more and shows better cytoplasmic distribution in the case of the lipopeptide AB36_siRNA complex compared to the HiPerFect_siRNA complex in HUVEC.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Flow Cytometry, Incubation, Microscopy, Staining, Sequencing

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: (a) Gene knockdown efficiency of the Notch1 gene by the lipopeptide_siRNA complex as compared to the HiPerFect_siRNA complex at 72 h transfection as evidenced by RT-PCR data. Lipopeptide AB18_siRNA complex shows slightly higher transfection efficacy as compared to the HiPerFect_siRNA complex at 72 h. (b) Immunofluorescence of Notch1 protein after transfection with the lipopeptide AB36_siRNA complex for 72 h compared to untreated cells, considered as a vehicle. Scale bar = 20 μM. (c) Long term gene knockdown efficiency of the lipopeptide AB36_siRNA complex compared to the HiPerFect_siRNA complex on day 3, day 7 and day 15 after the first transfection. On day 7, siRNA transporter AB36 exhibited 1.6 times more gene knockdown than HiPerFect, indicating its potential for long term gene silencing. (d) Relative expression of the epithelial marker, E-cadherin in Notch1 silenced MDA-MB-231 by the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex increased and (e) relative expression of the mesenchymal marker, N-cadherin in Notch1 silenced cells by the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex decreased. (f) Relative expression of the MMP-2 gene (responsible for lung metastasis) in Notch1 silenced MDA-MB-231 cells by the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex. In the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex treated Notch1 silenced MDA-MB-231 cells, the relative expression of (g) the CD44 gene declined and subsequently the expression of (h) the CD24 gene was increased with respect to untreated MDA-MB-231 indicating possible inhibition of stemness in the treated samples. (i and j) SEM images revealing nano-scale bridges connecting metastatic breast cancer MDA-MB-231 (epithelial) cells to HUVEC (endothelial cells) in the 3D co-culture. (i) Untreated MDA-MB-231 and (j) MDA-MB-231 cells treated with the lipopeptide AB36_Notch1 siRNA nanocomplex were co-cultured with HUVEC. Lipopeptide AB36_Notch1 silencing siRNA treated cells did not exhibit any nanobridge formation indicating the inhibition of metastasis compared to untreated cells taken as controls, which exhibited nanobridge connecting HUVEC cells (yellow arrows). (a and c–h) Data are represented as mean ± SEM of n = 3 at each data point. (a) and (c) *** p < 0.0001 and n.s. – not significant compared with untreated cells. The experiments were repeated twice. Lipopeptide AB36 (having the same peptide sequence like lipopeptide AB18) was used in (b), (c), (i) and (j) to avoid interference induced by FAM.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Expressing, Marker, Inhibition, Co-Culture Assay, Cell Culture, Sequencing

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: Knockdown percentage of oncogenes by different siRNA transporters

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Knockdown, Incubation

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: (a) Cytotoxicity analysis of MDA-MB-231 cells with the combination of different doses of metformin close to its IC50 and fixed dose of lipopeptide AB36_Notch1 siRNA for the determination of the combination index (CI) at 72 h. Data are represented as mean ± SEM of n = 3 at each data point (*** p < 0.0001, * p < 0.05 and n.s. – not significant compared with untreated cells). (b) CI vs. effect (Fa) at a non-fixed ratio. All of the drug combinations of drug doses are represented by blue dots (combination doses). Blue dots below CI = 1 represent synergistic interactions. (c) Relative expression of the MMP-2 gene in Notch1 silenced MDA-MB-231 cells by combination of metformin (200 μM) and the lipopeptide AB36_siRNA complex (MR 50) compared to monotherapy [ i.e. , separately by 200 μM metformin and also by lipopeptide AB36_Notch1 siRNA (MR 50)]. (d) Relative expression of the CD44 gene in Notch1 silenced MDA-MB-231 cells by combination of metformin (200 μM) and the lipopeptide AB36_siRNA complex (MR 50) compared to monotherapy. (e) Relative expression of the CD24 gene in Notch1 silenced MDA-MB-231 cells by combination of metformin (200 μM) and the lipopeptide AB36_siRNA complex (MR 50) compared to monotherapy. Combination reduces markers of metastasis (MMP-2) and stemness (CD44 and CD24) compared to monotherapy. (c–e) Data are represented as mean ± SEM of n = 3 at each data point. The experiments were repeated twice.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Expressing

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: In vivo zebrafish xenograft model for the evaluation of cell proliferation and micro-metastasis. (a) Day 0 images of zebrafish injected with untreated MDA-MB-231 cells at the left section, Notch1 silenced MDA-MB-231 cells with the lipopeptide AB36_Notch1 siRNA complex for 72 h at the centre and MDA-MB-231 cells treated with combination of metformin (200 μM) and the lipopeptide AB36_Notch1 siRNA complex (MR 50) for 72 h at the right section. (b) Images of zebrafish on day 5 post injection. The middle and tail portions were imaged with higher exposure for TRITC (red) for clear visualization of micro-metastasis and are inserted as an inset above each condition. Notch1 silenced MDA-MB-231 cells treated with the lipopeptide AB36_Notch1 siRNA complex and MDA-MB-231 cells treated with a combination of lipopeptide AB36_Notch1 siRNA and metformin (200 μM) showed significant reduction in cell proliferation and micrometastasis in the in vivo zebrafish model compared to untreated MDA-MB-231 cells. (c) Quantification of cell proliferation of MDA-MB-231 in the in vivo zebrafish xenograft model by measuring the fluorescence intensity of CM-Dil labelled MDA-MB-231 cells on the fifth day compared to the day of injection. Data are represented as mean ± SEM of n = 10 at each data point. The experiment was repeated twice. Quantification of fluorescence intensity was carried out using ImageJ software and is represented as % mean fluorescence intensity, where the reading of day 0 was taken as the baseline. Image stitching was also achieved using ImageJ software. (d) Graphical representation of the immunogenicity study calculating the stimulation index of peripheral blood mononuclear cells (PBMCs) isolated from goat blood by MTT studies. Designed lipopeptide AB36 and HiPerFect are non-immunogenic till 72 h. Data are represented as mean ± SEM of n = 3 at each data point. Lipopeptide AB36 (having a similar peptide sequence to lipopeptide AB18) was used to avoid interference induced by FAM.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: In Vivo, Injection, Fluorescence, Software, Immunopeptidomics, Isolation, Sequencing

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: 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: 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