Journal: bioRxiv

Article Title: ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN-response genes

doi: 10.1101/2020.09.02.279133

Figure Lengend Snippet: A-B) Western blot analysis to measure ETV7 protein expression in MCF7 (A) and T47D (B) cells stably over-expressing ETV7 or their empty control. GAPDH, Actinin and Tubulin expression were used as loading control. Blots were cropped for clarity and conciseness of the presentation. C-D) Cell Titer Glo Assay for survival analysis upon 5-FU treatment in MCF7 (C) and T47D (D) cells over-expressing ETV7 and their empty control. The percentage of viable cells was calculated normalizing the luminescence measures on the DMSO treated sample. E) Relative percentage of PI positive cells calculated as the difference of 5-FU and DMSO treated cells measured by Annexin V-FITC/PI staining of MCF7 Empty and MCF7 ETV7 cells treated with 5-FU 200 μM for 72 hours.33 F) RT-qPCR analysis of ABCB1, ABCC1, and ABCG2 expression in MCF7 Empty and MCF7 ETV7 cells. G) Western Blot analysis of the anti-apoptotic BCL-2 and Survivin protein expression in MCF7 Empty and MCF7 ETV7 cells. Tubulin was used as loading control. Bars represent the averages and standard deviations of at least three independent experiments. On the left of each blot is indicated the approximate observed molecular weight. * = p-value < 0.05; ** = p-value < 0.01.

Article Snippet: The expression vector pCMV6-Entry-ETV7 C-terminally tagged with DDK-Myc tags was purchased from Origene (Tema Ricerca, Bologna, Italy).

Techniques: Western Blot, Expressing, Stable Transfection, Glo Assay, Staining, Quantitative RT-PCR, Molecular Weight

Journal: bioRxiv

Article Title: ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN-response genes

doi: 10.1101/2020.09.02.279133

Figure Lengend Snippet: A-B) Colony formation/clonogenic assay of MCF7 (A) and T47D (B) Empty and ETV7 cells after 3 weeks of growth. Presented are a representative image (left) of the entire well and the total colony area (right), calculated with the ImageJ software. C-D) Soft agar colony formation assay of MCF7 (C) and T47D (D) Empty and ETV7 cells after 3 weeks of growth. On the left, a representative image of part of the well (taken at 5X magnification) and on the right the average colony area calculated with the ImageJ software. Indicated are the scale bars for microscopy images. Bars represent the averages and standard deviations of at least three independent experiment. *** = p-value < 0.001.

Article Snippet: The expression vector pCMV6-Entry-ETV7 C-terminally tagged with DDK-Myc tags was purchased from Origene (Tema Ricerca, Bologna, Italy).

Techniques: Clonogenic Assay, Software, Soft Agar Assay, Microscopy

Journal: bioRxiv

Article Title: ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN-response genes

doi: 10.1101/2020.09.02.279133

Figure Lengend Snippet: A-B) CD44-APC and CD24-FITC staining and flow cytometry analysis in MCF7 (A) and T47D (B) Empty and ETV7 cells. On the left, a representative dotplot of the results obtained at FACS Canto A; the histogram on the right summarizes the percentage of CD44+/CD24-cells in Empty and ETV7 over-expressing cells. C) A representative image of first-generation mammospheres obtained from MCF7 Empty and MCF7 ETV7 cells, respectively. The scale bar is indicated. D) The percentage of mammosphere formation efficiency (% MFE) in MCF7 Empty and ETV7 calculated as number of mammospheres per well/number of cells seeded per well X 100. E) % MFE in second-, third-, and fourth-generation mammospheres obtained by passaging the mammospheres every 7 days. Bars represent the averages and standard deviations of at least three independent experiments. * = p-value < 0.05; ** = p-value < 0.01; *** = p-value < 0.001.

Article Snippet: The expression vector pCMV6-Entry-ETV7 C-terminally tagged with DDK-Myc tags was purchased from Origene (Tema Ricerca, Bologna, Italy).

Techniques: Staining, Flow Cytometry, Expressing, Passaging

Journal: bioRxiv

Article Title: ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN-response genes

doi: 10.1101/2020.09.02.279133

Figure Lengend Snippet: A) A Venn diagram showing the number of differentially expressed genes (DEGs at a False Discovery Rate (FDR) ≤ 0.05) in the comparison between MCF7 and T47D cells over-expressing EVT7 and their respective controls. B-C) Gene Set Enrichment Analysis (GSEA) of MCF7 and T47D cells over-expressing ETV7 vs MCF7 and T47D controls. Enrichment plots for Type 1 Interferon response (B) and Type 2 Interferon response (C) gene sets of the Hallmark Collection. The Normalized Enrichment Score (NES) represents the degree of the enrichment of the gene set; the negative sign indicates that the gene set is down-regulated in cells over-expressing ETV7. D) Heatmaps showing the standardized expression level of genes comprising the ETV7-regulated IFN-responsive gene signature in MCF7 (left) and T47D (right) cells. E) RT-qPCR experiments for the validation of genes of the ETV7-regulated IFN-responsive signature with a Fold Change (FC) < −2 in MCF7 Empty and ETV7 cells. Bars represent the averages and standard deviations of at least three biological replicates. ** = p-value < 0.01; *** = p-value < 0.001. F) RT-qPCR analysis of normalized ETV7 expression relative to untreated control in MCF7 cells treated with 5ng/ml IFN-β (red) or IFN-γ (blue) at different time points. G) RT-qPCR analysis of the normalized expression of the genes regulated by ETV7 (IFI35, HERC6, APOL6) in MCF7 cells treated with 5ng/ml IFN-β (red) or IFN-γ (blue) at different time points. Bars represent the averages and SEM of at least three biological replicates.

Article Snippet: The expression vector pCMV6-Entry-ETV7 C-terminally tagged with DDK-Myc tags was purchased from Origene (Tema Ricerca, Bologna, Italy).

Techniques: Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN-response genes

doi: 10.1101/2020.09.02.279133

Figure Lengend Snippet: A-B) Percentage of first (A) and second (B) generation mammosphere formation efficiency (% MFE) in MCF7 Empty and ETV7 cells in response to 5ng/ml IFN-β or IFN-γ calculated as number of mammospheres per well/number of cells seeded per well X 100. C-D) Histograms summarizing the percentage of CD44 + /CD24 - cells measured by CD44-APC and CD24-FITC staining and flow cytometry analysis in MCF7 Empty and ETV7 cells (C) or T47D Empty and ETV7 cells (D) treated with 5ng/ml IFN-β and IFN-γ for 2 weeks. A representative dot plot of the results obtained at FACS Canto A in T47D cells is shown on the right. Bars represent the averages and standard deviations of at least three biological replicates. * = p-value < 0.05; ** = p-value < 0.01.

Article Snippet: The expression vector pCMV6-Entry-ETV7 C-terminally tagged with DDK-Myc tags was purchased from Origene (Tema Ricerca, Bologna, Italy).

Techniques: Staining, Flow Cytometry

Journal: bioRxiv

Article Title: ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN-response genes

doi: 10.1101/2020.09.02.279133

Figure Lengend Snippet: A, B) ETV7 expression in TCGA BRCA samples classified by tissue type (A) and PAM50 molecular subtype (B). Plot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Significance is calculated with ANOVA and post-hoc Tukey test. * = p-value < 0.05; *** = p-value < 0.001. C-F) Kaplan–Meier curves for TCGA breast cancer patients stratified according to the average expression of ETV7-regulated IFN-responsive gene signature. Curves represent the probability of disease specific survival (DSS) (C), overall survival (OS) (D), disease free interval (DSI) (E) and progression free interval (PFI) (F). p-values are calculated with logrank test.

Article Snippet: The expression vector pCMV6-Entry-ETV7 C-terminally tagged with DDK-Myc tags was purchased from Origene (Tema Ricerca, Bologna, Italy).

Techniques: Expressing

Journal: bioRxiv

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing TNFR1/NF-κB axis

doi: 10.1101/2022.09.06.506542

Figure Lengend Snippet: A-B) Gene Set Enrichment Analysis of MCF7 and T47D cells over-expressing ETV7 or its Empty counterpart. Enrichment plot for inflammatory response in MCF7 and T47D cells (A) and TNFA signalling via NFKB in MCF7 and T47D cells (B) gene sets of the Hallmark Collection. The Normalized Enrichment Score (NES) shows the degree of the enrichment of the gene set; the negative sign indicates that the gene set is down-regulated in cells over-expressing ETV7. FDR = False Discovery Rate. C) RT-qPCR analysis for the validation of genes involved in inflammation and immune response in MCF7 and T47D cells over-expressing ETV7 or Empty vector. Bars represent the averages and standard deviations of at least three biological replicates. D) The expression of TNFRSF1A at protein level in MCF7 and T47D cells over-expressing ETV7 or harbouring its empty counterpart. On the right of each blot is indicated the approximate observed molecular weight. HSP70 was used as a loading control. E) RT-qPCR analysis of the normalized expression of the TNFRSF1A gene in MDA-MB-231 and SK-BR-3 cells transiently over-expressing ETV7 or harbouring its Empty counterpart. Bars demonstrate the averages and standard deviations of at least three biological replicates. F) On the left a box plot demonstrating the differential expression analysis for the TNFRSF1A gene in a BRCA patients’ dataset. T= tumor (red), N=normal (grey), TPM=Transcripts per Million. GEPIA tool, based on TCGA (The Cancer Genome Atlas) databases, was used to obtain these data. On the right a box plot of TNFRSF1A expression in breast cancer patients when comparing normal and tumor RNA-seq data using TNM plot tool. Mann-Whitney test p value is shown in the right upper corner. Whole panel: * p≤0.05; ** p≤0.01; *** p≤0.001; n.s. – not significant.

Article Snippet: After 24 hours, cells were treated with 20 ng/ml IL-6 and 4 hours post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Expressing, Quantitative RT-PCR, Biomarker Discovery, Plasmid Preparation, Molecular Weight, Control, Quantitative Proteomics, RNA Sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing TNFR1/NF-κB axis

doi: 10.1101/2022.09.06.506542

Figure Lengend Snippet: A) A schematic view of the TNFRSF1A Intron 1 and the studied ETV7 binding sites. TNFRSF1A BS#1 is located +5,483bp form the Transcription Start Site (TSS); BS#2 +5,627bp from TSS; BS#3 +6,069bp from TSS. B-C) ChIP-qPCR of TNSFRSF1A Intron 1 in MCF7 (B) or T47D (C) cells over-expressing ETV7. The percentage of the enrichment of ETV7 or control (normal mouse IgG) bound to TNFRSF1A Intron 1 in respect to input DNA is shown. NSB=non-specific binding, the ACTB promoter. Bars represent the averages and standard deviations of at least three biological replicates. * p≤0.05; ** p≤0.01; *** p≤0.001, n.s. – not significant.

Article Snippet: After 24 hours, cells were treated with 20 ng/ml IL-6 and 4 hours post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Binding Assay, ChIP-qPCR, Expressing, Control

Journal: bioRxiv

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing TNFR1/NF-κB axis

doi: 10.1101/2022.09.06.506542

Figure Lengend Snippet: A-B) Gene reporter assays in MCF7 (A) and T47D (B) cells over-expressing ETV7, or its empty counterpart transiently transfected with pGL3-NF-κB reporter plasmid. Data is normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty control. C-D) Gene reporter assays in MCF7 (C) and T47D (D) cells over-expressing ETV7, or its empty counterpart transfected with pGL3-NF-κB reporter plasmid and stimulated with TNF-α (10 ng/ml for MCF7 and 15 ng/ml for T47D), IL-6 (20 ng/ml) or combination of both for 4 hours. Data is normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty control. F-I) RT-qPCR analysis of known NF-κB target genes: A20 (E), TNF-α (F), IL-8 (G) and IL-6 (H) in MCF7 ETV7 or Empty cells treated with TNF-α (10 ng/ml) for 4 hours. Bars represent the averages and standard deviations of at least three biological replicates. I) Western blot analysis of phosphorylated IκBα in MCF7 Empty and ETV7 cells in response to 10 ng/ml TNF-α treatment for 1 hours. On the right of each blot is indicated the approximate observed molecular weight. GAPDH was used as a loading control. J) A flowchart of the design of the rescue experiment. K) Gene reporter assays in MCF7 Empty or MCF7 ETV7 cells transfected with pGL3-NF-κB reporter vector and pcDNA3.1-TNFR1 or pcDNA3.1-Empty plasmids and untreated or treated with 10 ng/ml TNF-α for 4 hours. Data is normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty untreated control. Whole panel: * p≤0.05; ** p≤0.01; *** p≤0.001.

Article Snippet: After 24 hours, cells were treated with 20 ng/ml IL-6 and 4 hours post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Control, Quantitative RT-PCR, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing TNFR1/NF-κB axis

doi: 10.1101/2022.09.06.506542

Figure Lengend Snippet: A) Canonical ETV7 and STAT3 binding sites known from the literature. B) Gene Set Enrichment Analysis of MCF7 cells over-expressing ETV7 or its Empty counterpart. Enrichment plot for IL6_JAK_STAT3 signalling in MCF7 cells gene sets of the Hallmark Collection. The Normalized Enrichment Score (NES) shows the degree of the enrichment of the gene set; the negative sign indicates that the gene set is down-regulated in cells over-expressing ETV7. FDR = False Discovery Rate. C) Western blot analysis of subcellular fractionation from MCF7 Empty and MCF7 ETV7 cells untreated or treated with IL-6 (20 ng/ml) for 4 hours. On the right of each blot is indicated the approximate observed molecular weight. Cyt – cytoplasmatic protein fraction, Chr – chromatin-enriched protein fraction. GAPDH was used as a loading control for the cytoplasmatic fraction. Histone H3 was used as a loading control for chromatin-enriched protein fraction. D-E) ChIP-qPCR of TNSFRSF1A Intron 1 Binding site #1 (D) and Binding site #2 (E) in MCF7 cells over-expressing ETV7 untreated or treated with IL-6 (20 ng/ml) for 4 hours. The percentage of the enrichment of pSTAT3 or control (normal rabbit IgG) bound to TNFRSF1A Intron 1 in respect to Input DNA is shown. NSB=non-specific binding, a region within the ACTB promoter. BS = binding site. Bars represent the averages and standard deviations of at least three biological replicates. F-G) Gene reporter assays in MCF7 Empty/ETV7 (F) and T47D Empty/ETV7 (G) cells transfected with M67-STAT3 reporter untreated or treated with IL-6 (20 ng/ml) for 4 hours. Data is normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty untreated control. Whole panel: * p≤0.05; ** p≤0.01; *** p≤0.001.

Article Snippet: After 24 hours, cells were treated with 20 ng/ml IL-6 and 4 hours post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Binding Assay, Expressing, Western Blot, Fractionation, Molecular Weight, Control, ChIP-qPCR, Transfection, Luciferase, Plasmid Preparation

Journal: bioRxiv

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing TNFR1/NF-κB axis

doi: 10.1101/2022.09.06.506542

Figure Lengend Snippet: A) The canonical STAT3/TNF-α/NF-κB regulatory pathway. STAT3 binds to its regulatory element in the first intron of the TNFRSF1A gene and induce its expression. Consequently, the TNF-α receptor 1 is produced. TNF-α molecules bind TNFR1 receptor and activates NF-κB signalling pathway. B) In the context where ETV7 expression is increased, ETV7 can displace STAT3 from its binding sites on the Intron 1 of TNFRSF1A and directly repress its expression. This ETV7-mediated repression leads to the reduced activation of NF-κB signalling and, hence, reduces the expression of pro-inflammatory genes.

Article Snippet: After 24 hours, cells were treated with 20 ng/ml IL-6 and 4 hours post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Expressing, Produced, Binding Assay, Activation Assay