anti v5 mouse monoclonal antibody  (R&D Systems)

Journal: Journal of Ovarian Research

Article Title: Biological and prognostic value of ETV5 in high-grade serous ovarian cancer

doi: 10.1186/s13048-021-00899-6

Figure Lengend Snippet: qRT-PCR relatively primer sequences

Article Snippet: The primary antibodies used in this study included mouse anti-human monoclonal ETV5 antibody (1:1000 dilution; Biorbyt, UK) and mouse anti-human monoclonal beta (β)-actin antibody (1:1000 dilution; Zhongshan Biotechnology, Beijing, China).

Techniques:

Journal: Journal of Ovarian Research

Article Title: Biological and prognostic value of ETV5 in high-grade serous ovarian cancer

doi: 10.1186/s13048-021-00899-6

Figure Lengend Snippet: ETV5 was highly expressed in HGSOC tissues and cell lines. ( A ) Overall, 64 differentially expressed genes between ovarian carcinoma and normal tissues were screened from 3 datasets. ( B) In GEPIA, ETV5 was highly expressed in ovarian carcinoma compared to that in normal tissues ( p < 0.05). ( C ) ETV5 was significantly overexpressed in HGSOC tissues compared to that in normal fallopian tube tissues ( p < 0.05). ( D ) ETV5 was relatively high expressed in HGSOC cell lines. Three ETV5-knockdown plasmids were transfected in SKOV3 ( E ) and A2780 ( F ) cell lines to screen out the plasmid with the best knockdown efficiency-sh-ETV5–2. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The primary antibodies used in this study included mouse anti-human monoclonal ETV5 antibody (1:1000 dilution; Biorbyt, UK) and mouse anti-human monoclonal beta (β)-actin antibody (1:1000 dilution; Zhongshan Biotechnology, Beijing, China).

Techniques: Knockdown, Transfection, Plasmid Preparation

Journal: Journal of Ovarian Research

Article Title: Biological and prognostic value of ETV5 in high-grade serous ovarian cancer

doi: 10.1186/s13048-021-00899-6

Figure Lengend Snippet: ETV5 expression and clinicopathological parameters

Article Snippet: The primary antibodies used in this study included mouse anti-human monoclonal ETV5 antibody (1:1000 dilution; Biorbyt, UK) and mouse anti-human monoclonal beta (β)-actin antibody (1:1000 dilution; Zhongshan Biotechnology, Beijing, China).

Techniques: Expressing, Over Expression

Journal: Journal of Ovarian Research

Article Title: Biological and prognostic value of ETV5 in high-grade serous ovarian cancer

doi: 10.1186/s13048-021-00899-6

Figure Lengend Snippet: ETV5 is associated with a poor prognosis of HGSOC patients. In comparison to patients with low ETV5 expression, those with high expression has significantly lower ( A ) overall survival (OS) ( p < 0.05) and ( B ) disease-free survival (DFS) ( p < 0.05)

Article Snippet: The primary antibodies used in this study included mouse anti-human monoclonal ETV5 antibody (1:1000 dilution; Biorbyt, UK) and mouse anti-human monoclonal beta (β)-actin antibody (1:1000 dilution; Zhongshan Biotechnology, Beijing, China).

Techniques: Comparison, Expressing

Journal: Journal of Ovarian Research

Article Title: Biological and prognostic value of ETV5 in high-grade serous ovarian cancer

doi: 10.1186/s13048-021-00899-6

Figure Lengend Snippet: COX regression analysis of risk factors in patients with high-grade serous ovarian carcinoma

Article Snippet: The primary antibodies used in this study included mouse anti-human monoclonal ETV5 antibody (1:1000 dilution; Biorbyt, UK) and mouse anti-human monoclonal beta (β)-actin antibody (1:1000 dilution; Zhongshan Biotechnology, Beijing, China).

Techniques: Expressing

Journal: Journal of Ovarian Research

Article Title: Biological and prognostic value of ETV5 in high-grade serous ovarian cancer

doi: 10.1186/s13048-021-00899-6

Figure Lengend Snippet: ETV5 promotes HGSOC cell proliferation, invasion, and migration in vitro. Cell proliferation is determined using the CCK8 assay after SKOV3 ( A ) and A2780 ( B ) cells are transfected with sh-ETV5–2 plasmid and OV2008 ( C ) cells are transfected with ETV5 plasmid. Clonality is detected using the colony formation assay after SKOV3 ( D ) and A2780 ( E ) cells are transfected with sh-ETV5–2 plasmid and OV2008 ( I ) cells. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The primary antibodies used in this study included mouse anti-human monoclonal ETV5 antibody (1:1000 dilution; Biorbyt, UK) and mouse anti-human monoclonal beta (β)-actin antibody (1:1000 dilution; Zhongshan Biotechnology, Beijing, China).

Techniques: Migration, In Vitro, CCK-8 Assay, Transfection, Plasmid Preparation, Colony Assay

Journal: Scientific reports

Article Title: Dynamics of chromatin accessibility during TGF-β-induced EMT of Ras-transformed mammary gland epithelial cells.

doi: 10.1038/s41598-017-00973-4

Figure Lengend Snippet: Figure 4. Global analysis of Etv4 and Etv5 binding regions in the genome. (A) Expression of Etv4 and Etv5 by RNA-seq of EpH4 and EpRas cells treated with 1 ng/ml TGF-β for 48 h. FPKM, fragments per kilobase of exons per million mapped fragments. The experiment was performed in two biological replicates. Data are a subset of the findings shown in Supplementary Fig. S2C. (B) Immunoblot analysis of endogenous Etv4 protein expression in EpH4 and EpRas cells. pSmad2, phosphorylated Smad2. (C) Relationship between the anti-Etv4 and anti-Etv5 ChIP-seq data and FAIRE-seq data obtained from TGF-β-treated EpRas cells. (D) De novo motif prediction identifies ETS family (ELK4) binding motif, and shown as in Fig. 3A. Anti-Etv4 ChIP-seq data were used for enriched motif calculation. q value (minimal false discovery rate required to include the motif) is shown. (E) Etv4 and Etv5 binding regions at the Cdh1 and Fn1 gene loci. HBB cluster region is shown as a control. Black bars represent the significant Etv4 and Etv5 binding regions. Significant FAIRE-positive regions in EpH4 and EpRas cells, without (first row) and with (second row) TGF-β stimulation, are shown as blue and red bars, respectively, as a reference (shown in Fig. 2A).

Article Snippet: Rabbit polyclonal anti-Etv4 antibody, previously used for ChIP-seq by Hollenhorst et al.54, was from AVIVA Systems Biology (ARP32263, San Diego, CA, USA), and mouse monoclonal anti-Etv5 antibody (MAB7107) was from R&D Systems.

Techniques: Binding Assay, Expressing, RNA Sequencing, Western Blot, ChIP-sequencing, Control

Journal: Scientific reports

Article Title: Dynamics of chromatin accessibility during TGF-β-induced EMT of Ras-transformed mammary gland epithelial cells.

doi: 10.1038/s41598-017-00973-4

Figure Lengend Snippet: Figure 5. Effect of Etv4 and Etv5 siRNAs on stress fiber formation, cell invasiveness and global gene expression in EpRas cells. (A) Effect of the siRNAs on the expression of Etv4 protein. EpRas cells were transfected with the siRNAs as indicated. After 24 h of incubation, cells were treated with TGF-β for additional 48 h and lysed for immunoblotting analysis. (B) Effect of Etv4/5 siRNAs on the TGF-β-induced down-regulation of E-cadherin protein. siRNA-transfected cells were treated with TGF-β for 8 days. Transfection of siRNAs were repeated at day 2 and 5. (C) Effect of Etv4/5 siRNAs on the stress fiber formation induced by TGF-β. F-actin formation of EpRas cells was evaluated by phalloidin staining. Cells were transfected with siRNAs for Etv4/5 and stimulated with TGF-β for 48 h. (D) Matrigel invasion assay in EpRas cells. Cells were transfected with the indicated siRNAs and then seeded on the Matrigel-coated plate, incubated for 48 h with TGF-β, and fixed. (upper panels) Representative images of the cells that migrated through the Matrigel-coated membrane. (bottom graph) Quantified data representing the means of four independent experiments. Error bars, standard deviations. (E and F) EpRas cells were transfected with siRNAs for Etv4/5 and treated with 1 ng/ml TGF-β for 48 h. Then, RNA-seq and ontology analysis using GSEA were performed. Genes with maximum FPKM (fragments per kilobase of exon per million mapped sequence reads) values ≥5 among the samples were selected for evaluation. (E) A list of top-enriched c5 (gene ontology) gene sets (MSigDB) downregulated by siEtv4/5 in the absence of TGF-β. The averaged gene expression data from the two distinct sets of siEtv4/5-transfected cells were compared to the data from the siNC-transfected cells. SIZE: number of genes in the phenotype, NES: normalized enrichment score, FDR: false discovery rate. (F) An enrichment plot showing the up-regulation of a gene set “EXTRACELLULAR REGION” by Etv4 and Etv5, which was identified as the top-enriched phenotype by GSEA.

Article Snippet: Rabbit polyclonal anti-Etv4 antibody, previously used for ChIP-seq by Hollenhorst et al.54, was from AVIVA Systems Biology (ARP32263, San Diego, CA, USA), and mouse monoclonal anti-Etv5 antibody (MAB7107) was from R&D Systems.

Techniques: Gene Expression, Expressing, Transfection, Incubation, Western Blot, Staining, Invasion Assay, Membrane, RNA Sequencing, Sequencing

Journal: Scientific reports

Article Title: Dynamics of chromatin accessibility during TGF-β-induced EMT of Ras-transformed mammary gland epithelial cells.

doi: 10.1038/s41598-017-00973-4

Figure Lengend Snippet: Figure 6. Regulation of Mmp13 expression by Etv4 and Etv5. (A) FAIRE-seq and ChIP-seq data at the matrix metalloproteinase 13 (Mmp13) gene locus as a target of Etv4 and 5. Data are shown as in Figs 2A and 4E. Arrow heads show the positions evaluated by FAIRE-qPCR in (D). (B) RNA-seq data of siEtv4/5-transfected EpRas cells treated with TGF-β. siNC; negative control siRNA. Error bars, 95% confidence intervals. (C) Matrigel invasion assay in EpRas cells. Cells were transfected with the indicated siRNAs and then seeded on the Matrigel- coated plate, incubated for 48 h with TGF-β, and fixed. (upper panels) Representative images of the cells that migrated through the Matrigel-coated membrane. (bottom graph) Quantified data representing the means of four independent experiments. Error bars, standard deviations. (D) Changes in the FAIRE-seq signal intensities by Etv4/5 siRNAs at FAIRE-positive regions of Mmp13 gene locus shown in (A). EpRas cells transfected with the indicated siRNAs were treated with TGF-β for 4 days and fixed for FAIRE-seq data acquisition. The data represent the result of two biological replicates. Error bars, standard deviations.

Article Snippet: Rabbit polyclonal anti-Etv4 antibody, previously used for ChIP-seq by Hollenhorst et al.54, was from AVIVA Systems Biology (ARP32263, San Diego, CA, USA), and mouse monoclonal anti-Etv5 antibody (MAB7107) was from R&D Systems.

Techniques: Expressing, ChIP-sequencing, RNA Sequencing, Transfection, Negative Control, Invasion Assay, Incubation, Membrane

Journal: Scientific reports

Article Title: Dynamics of chromatin accessibility during TGF-β-induced EMT of Ras-transformed mammary gland epithelial cells.

doi: 10.1038/s41598-017-00973-4

Figure Lengend Snippet: Figure 7. Effect of Etv4/5 siRNAs on the global chromatin accessibility. (A) Scatter plots showing the effect of TGF-β (X-axis) and Etv4/5 siRNAs (Y-axis) on the genome-wide chromatin accessibility determined by FAIRE-seq. Note that there was a significant correlation between the effect of two different siRNA sets (r = 0.51, p < 2.2e-16, Supplementary Fig. S7). (B) Enrichment of ETS binding motif in the FAIRE-positive regions of EpRas cells treated by TGF-β and siRNAs as indicated. (C) A schematic model for regulation of EMT-related gene expression through the differential expression of transcription factors and alteration of chromatin accessibility. In the process of EMT, chromatins are gradually closed by Ras and TGF-β at the Cdh1/E-cadherin gene locus. In contrast, upregulated genes are regulated by a variety of mechanisms. The Fn1/fibronectin gene locus is open irrespective of Ras and TGF-β treatment, while Cdh2/N-cadherin gene locus becomes accessible upon Ras transformation, and the Mmp13 gene locus becomes accessible upon Ras transformation and TGF-β stimulation. Differentially expressed transcription factors, Etv4 and Etv5, are responsible for a subset of EMT- related changes in gene expression partially through the alteration of chromatin accessibility.

Article Snippet: Rabbit polyclonal anti-Etv4 antibody, previously used for ChIP-seq by Hollenhorst et al.54, was from AVIVA Systems Biology (ARP32263, San Diego, CA, USA), and mouse monoclonal anti-Etv5 antibody (MAB7107) was from R&D Systems.

Techniques: Genome Wide, Binding Assay, Gene Expression, Quantitative Proteomics, Transformation Assay