human etv1  (Addgene inc)

Journal: bioRxiv

Article Title: FUS controls muscle differentiation and structure through LLPS mediated recruitment of MEF2 and ETV5

doi: 10.1101/2024.09.18.613669

Figure Lengend Snippet: FUS is a transcriptional co-activator of PEA3 transcription factors a: Pie chart showing the distribution of FUS binding sites identified by ChIP-seq analysis in C2C12 cells. FUS binds mostly to promoters, close to the transcription start sites (TSS). b-c: Mean profiles established for FUS binding at TSS using SeqMiner in 3 independent replicates. d: Genome browser visualization of FUS binding on the promoter region of the Mrps18c and Helq genes. e: Gene ontology analysis of genes with FUS binding sites identified by ChIP-seq in C2C12 cells. f-g: MEME motif enriched in ChIP-seq peaks bound by FUS (F) and the predicted motif bound by PEA3 transcription factor ETV4 (G). h-j: Light units relative to empty control plasmid in C2C12 cells 24 h after transfection of an Mrps18c- luciferase plasmid and an expression plasmid for either hFUS WT, hFUS R495X or empty control. Cells were simultaneously transfected with si Ctr (25 nM) or si ETV1 (25 nM, H), si ETV4 (25 nM, I) and si ETV5 (25nM, J). Nested one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001, **P<0.01, *P < 0.05. Each dot represents the mean of an individual experiment (n=3), each consisting of 4 technical replicates. Data represent the mean ± SEM. k: Light units relative to si Ctr in C2C12 cells 24 h after transfection of Mrps18c- luciferase plasmid and si Ctr (25 nM) or si FUS and an expression plasmid for either ETV5 or empty control. Nested one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001. Each dot represents the mean of an individual experiment (n=3), each consisting of 4 technical replicates. Data represent the mean ± SEM.

Article Snippet: ETV1 (# TP310533), ETV4 (# TP760035), ETV5 (# TP761784) and MEF2A (# TP312830) were purchased from Origen.

Techniques: Binding Assay, ChIP-sequencing, Control, Plasmid Preparation, Transfection, Luciferase, Expressing

Journal: bioRxiv

Article Title: FUS controls muscle differentiation and structure through LLPS mediated recruitment of MEF2 and ETV5

doi: 10.1101/2024.09.18.613669

Figure Lengend Snippet: a: Genome browser snapshot of MEF2A, ETV5 and FUS ChIP-seq binding sites at the MRPS18C/HELQ locus. b: Venn diagram showing the overlap between FUS, MEF2A and ETV5 binding sites. Hypergeometric p-values: Fus vs Etv5: 3.66×10 -2 ; Fus vs Mef2a: <5×10 -324 ; Etv5 vs Mef2a: <5×10 -324 . c-e: Light units relative to Empty vector + si Ctr in C2C12 cells 24 h after transfection of a 3xMEF2 luciferase plasmid and an expression plasmid for either hFUS WT, hFUS R495X or empty control. Cells were simultaneously transfected with si Ctr (25 nM) or si ETV1 (25 nM, C), si ETV4 (25 nM, D), and si ETV5 (25nM, E). ETV5, but not ETV1 or ETV4, is required for co-activation of the MEF2 reporter by FUS. Nested one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001, **P<0.01, *P < 0.05. Each dot represents the mean of an individual experiment (n=4), each consisting of 4 technical replicates. Data represent the mean ± SEM.

Article Snippet: ETV1 (# TP310533), ETV4 (# TP760035), ETV5 (# TP761784) and MEF2A (# TP312830) were purchased from Origen.

Techniques: ChIP-sequencing, Binding Assay, Plasmid Preparation, Transfection, Luciferase, Expressing, Control, Activation Assay

Journal: OncoTargets and therapy

Article Title: miR-129-5p inhibits prostate cancer proliferation via targeting ETV1

doi: 10.2147/OTT.S183435

Figure Lengend Snippet: ETV1 was negatively regulated by miR-129-5p. Notes: ( A ) In comparison with normal epithelial prostate cell line RWPE-1, expression of ETV1 mRNA was reduced in prostate cancer cell lines (PC-3, DU145, and LNCaP). ( B ) MiR-129-5p mimics decreased ETV1 mRNA expression in PC-3 cells. ( C ) MiR-129-5p mimics decreased ETV1 protein expression in PC-3 cells. ( D ) Quantitative analysis of ETV1 protein expression in ( C ). ( E ) MiR-129-5p antagonist increased ETV1 mRNA expression in RWPE-1 cells. ( F ) MiR-129-5p antagonist increased ETV1 protein expression in RWPE-1 cells. ( G ) Quantitative analysis of ETV1 protein expression in ( F ). * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Full length of ETV1 open reading frame was amplified from RWPE-1 cDNA and ligated into pcDNA3.1 plasmid (OriGene, Rockville, MD, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: miR-129-5p inhibits prostate cancer proliferation via targeting ETV1

doi: 10.2147/OTT.S183435

Figure Lengend Snippet: ETV1 was a direct target of miR-129-5p. Notes: ( A ) Sequence alignment of ETV1 mRNA 3′UTR-WT (wild type), 3′UTR-Mut (mutant), and miR-129-5p sequence. ( B ) Dual Luciferase Reporter Assay showed that miR-129-5p mimics reduced luciferase activity of PC-3 cells transfected with pGL3-ETV1 3′UTR-WT not mutant ETV1 3′UTR. ( C ) Dual Luciferase Reporter Assay showed that miR-129-5p mimics reduced luciferase activity of RWPE-1 cells transfected with pGL3-ETV1 3′UTR-WT not mutant ETV1 3′UTR. ** P <0.01.

Article Snippet: Full length of ETV1 open reading frame was amplified from RWPE-1 cDNA and ligated into pcDNA3.1 plasmid (OriGene, Rockville, MD, USA).

Techniques: Sequencing, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Transfection

Journal: OncoTargets and therapy

Article Title: miR-129-5p inhibits prostate cancer proliferation via targeting ETV1

doi: 10.2147/OTT.S183435

Figure Lengend Snippet: Forced elevation of ETV1 expression rescued cell growth arrest and inhibition of colony forming ability induced by miR-129-5p mimics in PC-3 cells. Notes: ( A ) MiR-129-5p mimics decreased ETV1 protein level, which could be reversed by transfection of pcDNA3.1-ETV1. ( B ) Quantitative analysis of ETV1 protein level in ( A ). ( C ) Overexpression of ETV1 attenuated the suppression effect of miR-129-5p mimics on cell growth. ( D ) In a colony forming assay, ETV1 overexpression attenuated the decrease of colony number induced by miR-129-5p mimics. ( E ) Quantitative analysis of colony number in ( D ). * P <0.05, ** P <0.01, *** P <0.001. Abbreviation: CCK-8, Cell Counting Kit-8.

Article Snippet: Full length of ETV1 open reading frame was amplified from RWPE-1 cDNA and ligated into pcDNA3.1 plasmid (OriGene, Rockville, MD, USA).

Techniques: Expressing, Inhibition, Transfection, Over Expression, CCK-8 Assay, Cell Counting

Journal: OncoTargets and therapy

Article Title: miR-129-5p inhibits prostate cancer proliferation via targeting ETV1

doi: 10.2147/OTT.S183435

Figure Lengend Snippet: Expression of miR-129-5p was inversely associated with ETV1 mRNA levels in prostate cancer tissues. Notes: ( A ) Representative Western blot of ETV1 expression in three pairs of prostate tumor tissues and matched normal tissues. ( B ) Quantification of ETV1 protein levels in prostate tumor tissues and matched normal tissues in ( A ). ( C ) Reverse transcription-quantitative PCR showed that ETV1 mRNA levels were significantly elevated in 30 prostate tumor tissues compared with adjacent normal prostate tissues. ( D ) Analysis of miR-129-5p expression and ETV mRNA levels showed a negative correlation between them. ( E ) Diagram of an miR-129-5p/ETV1/Hippo-YAP axis in prostate cancer cells. * P <0.05, *** P <0.001.

Article Snippet: Full length of ETV1 open reading frame was amplified from RWPE-1 cDNA and ligated into pcDNA3.1 plasmid (OriGene, Rockville, MD, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells

doi: 10.1016/j.isci.2022.105402

Figure Lengend Snippet:

Article Snippet: ETV1 Human qPCR Primer Pair , OriGene , cat# HP208189.

Techniques: Recombinant, Transfection, Knock-Out, SYBR Green Assay, Derivative Assay, Plasmid Preparation, Software

Journal: iScience

Article Title: Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells

doi: 10.1016/j.isci.2022.105402

Figure Lengend Snippet: Evaluation of expression of key Purkinje markers in control vs PURK-cocktail-treated cells Immunocytochemistry and fluorescent microscopy showed that the control cells did not express key Purkinje markers [ETV1, IRX3, SCN5a, or PCP4] or the CRISPR-Cas9 Knock-in CNTN2-IRES-mCherry. In contrast, the PURK-cocktail-treated cells strongly expressed ETV1, IRX3, SCN5a, or PCP4 and the CRISPR-Cas9 Knock-in CNTN2-IRES-mCherry reporter gene. DAPI was used to stain the cells' nuclei. The scale bar corresponds to 100 μm in all pictures.

Article Snippet: ETV1 Human qPCR Primer Pair , OriGene , cat# HP208189.

Techniques: Expressing, Control, Immunocytochemistry, Microscopy, CRISPR, Knock-In, Staining

Journal: iScience

Article Title: Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells

doi: 10.1016/j.isci.2022.105402

Figure Lengend Snippet: qPCR data evaluating the expression of key Purkinje markers in control and PURK-cocktail-treated cells The PURK-cocktail treatment induced a different genetic profile, which resembled closely to that of native cardiac Purkinje cells on both AC16-CM and iPSC-CM compared to the control. Data corresponds to mean ± SEM of n=3. ∗,∗∗,∗∗∗,∗∗∗∗ corresponds to p < 0.05, 0.01, 0.001, and 0.0001 in a two-Way ANOVA test. “ns” corresponds to “not significant.”

Article Snippet: ETV1 Human qPCR Primer Pair , OriGene , cat# HP208189.

Techniques: Expressing, Control

Journal: iScience

Article Title: Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells

doi: 10.1016/j.isci.2022.105402

Figure Lengend Snippet:

Article Snippet: ETV1 Human qPCR Primer Pair , OriGene , cat# HP208189.

Techniques: Recombinant, Transfection, Knock-Out, Multiplex sample analysis, SYBR Green Assay, Reverse Transcription, Derivative Assay, Plasmid Preparation, Software

Journal: iScience

Article Title: Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells

doi: 10.1016/j.isci.2022.105402

Figure Lengend Snippet: List of antibodies used in immunocytochemistry experiments

Article Snippet: ETV1 Human qPCR Primer Pair , OriGene , cat# HP208189.

Techniques: Immunocytochemistry

Journal: iScience

Article Title: Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells

doi: 10.1016/j.isci.2022.105402

Figure Lengend Snippet: qPCR program

Article Snippet: ETV1 Human qPCR Primer Pair , OriGene , cat# HP208189.

Techniques: Activation Assay

Journal: eLife

Article Title: Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer

doi: 10.7554/eLife.66524

Figure Lengend Snippet: ( a ) Upregulated genes by KRAS G12V overexpression for 1 day and 7 days in comparison with a GFP overexpression control in H2107 and H82 cells. The numbers of genes upregulated (>1.5 fold) are indicated. ETV1 , ETV4 , and ETV5 are shown in red. ( b ) Heat map of the PEA3 family ETS transcription factors ( ETV1 , ETV4 , and ETV5 ) and neuroendocrine transcription factors ( ASCL1 , NEUROD1 , INSM1 , and POU3F2 [ BRN2 ]) in small cell lung cancer and lung adenocarcinoma cell lines. Red and blue denote high and low expression, respectively. ( c ) Western blot showing the effects of ERK inhibition using 1 μM SCH772984 on the expression of ETV1, ETV4, and ETV5 with or without KRAS G12V transduction for 72 hr. Lysates from HA-tagged ETV1-overexpressing H524 cells were used as a positive control for ETV1. GAPDH was used as a loading control. ( d ) Effects of HA-tagged ETV1 induction as assessed by western blot in H2107, H82, and H524 cells, upon treatment with 100 ng/mL doxycycline (dox) for 3 and 7 days. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated control values (set to two in each panel) after normalization to GAPDH. ( e ) Effects of HA-tagged ETV5 induction as assessed by western blot in H82 and H524 cells, upon treatment with 100 ng/mL dox for 3 and 7 days. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated control values (set to one in each cell line panel) after normalization to GAPDH. ( f ) Western blot showing the effects of KRAS G12V induction and treatment with siRNA pools targeting ETV4 , ETV5 , or both on expression of neuroendocrine transcription factors in H82 cells. Cells were treated with 100 ng/mL dox and indicated siRNAs for 72 hr. Scrambled siRNA (siScr) was used as a negative control. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated and siScr-treated control values (set to 1) after normalization to GAPDH. ( g ) Western blot showing the effects of MSK/RSK and/or CBP/p300 inhibition on KRAS G12V -mediated expression of ETV4 and ETV5 in H82 cells. Cells were treated with 5 μM SB-747651A (a MSK-RSK inhibitor) and/or 400 nM A-485 (a CBP/p300 inhibitor) as well as 100 ng/mL dox for 72 hr. GAPDH was used as a loading control. Numbers below the ETV5 blots indicate the amounts of ETV5 relative to the dox-treated and non-drug-treated (DMSO-treated) control values (set to lane 5) after normalization to GAPDH. Immunoblots are representative of at least two biological replicates.

Article Snippet: Human ETV1 (Addgene, Cambridge, MA, USA; plasmid #82209) and ETV5 (Horizon Discovery, Cambridge, UK; clone 100008315) were transferred to pInducer20 using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific).

Techniques: Over Expression, Comparison, Control, Expressing, Western Blot, Inhibition, Transduction, Positive Control, Negative Control

Journal: eLife

Article Title: Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer

doi: 10.7554/eLife.66524

Figure Lengend Snippet: ( a ) Western blot of RB in RB1 -proficient parental PC9 and H1975 cells as well as RB1 -knockout polyclonal and clonal cells. GAPDH was used as a loading control. ( b ) Western blot showing effects of 100 nM osimertinib treatment for up to 5 days on protein expression of neuroendocrine transcription factors and SOX2 in RB1 -proficient and -deficient PC9 and H1975 cells. Lysates from the small cell lung cancer cell line, H2107 or H82, were used as a positive control for neuroendocrine markers. Lysates from H2107 were used as a positive control for SOX2. GAPDH was used as a loading control. ( c ) Mean relative proliferation of parental PC9 and H1975 cells with or without RB1 knockout treated with osimertinib. Cells were treated with osimertinib or DMSO for 72 hr. Bars represent mean (three biological replicates) ± SD. The IC 50 values for each clone are as follows: PC9-sgControl, 7.0 nM; PC9-sg RB1 #1, 5.9 nM; PC9-sg RB1 #15, 4.8 nM; H1975-sgControl, 19 nM; H1975-sg RB1 #9, 145 nM; and H1975-sg RB1 #22, 57 nM. IC 50 analysis of dose-response curves were compared by the extra sum-of-squares F test. ( d ) Mean relative proliferation of PC9 and H1975 cells with acquired resistance to osimertinib are plotted. Osimertinib-resistant RB1 -proficient cells as well as RB1 -knockout clonal cells were treated with osimertinib for 72 hr. Control cells were treated with DMSO as a vehicle control. Osimertinib-resistant cells were generated by either a stepwise dose-escalation or initial high-dose method. Bars represent mean (three biological replicates) ± SD. ( e ) Profiling of acquired genetic alterations through osimertinib treatment in PC9 and H1975 cells with or without RB1 knockout assessed by MSK-IMPACT. Abbreviations: stepwise, stepwise dose-escalation method; initial, initial high-dose method. ( f ) Western blot for profiling expression of EGFR, ERK, RAS, and neuroendocrine factors in parental and osimertinib-resistant PC9 and H1975 cells with or without RB1 knockout. Parental and resistant cells were harvested under treatment with 0.1% DMSO or osimertinib (2 μM for H1975 [stepwise dose-escalation method] and 1 μM for the others), respectively. Lysates from the small cell lung cancer cell line, H2107 or H82, were used as a positive control for neuroendocrine markers. GAPDH was used as a loading control. ( g ) Western blot for profiling expression of CIC as well as its downstream targets ETV1, ETV4, and ETV5 in parental and osimertinib-resistant H1975 cells. Parental cells were treated with 100 nM osimertinib or DMSO for 3 days. Osimertinib-resistant cells were cultured with 2 μM (stepwise dose-escalation method) or 1 μM (initial high-dose method) osimertinib. GAPDH was used as a loading control.

Article Snippet: Human ETV1 (Addgene, Cambridge, MA, USA; plasmid #82209) and ETV5 (Horizon Discovery, Cambridge, UK; clone 100008315) were transferred to pInducer20 using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific).

Techniques: Western Blot, Knock-Out, Control, Expressing, Positive Control, Generated, Cell Culture

Journal: eLife

Article Title: Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer

doi: 10.7554/eLife.66524

Figure Lengend Snippet:

Article Snippet: Human ETV1 (Addgene, Cambridge, MA, USA; plasmid #82209) and ETV5 (Horizon Discovery, Cambridge, UK; clone 100008315) were transferred to pInducer20 using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Mutagenesis, Recombinant, Sequencing, Control, Gene Expression, Extraction, Software