Journal: International Journal of Molecular Sciences

Article Title: Mitigation of Breast Cancer Cells’ Invasiveness via Down Regulation of ETV7, Hippo, and PI3K/mTOR Pathways by Vitamin D3 Gold-Nanoparticles

doi: 10.3390/ijms25105348

Figure Lengend Snippet: ( A ) Representative Western blots of PI3K, AKT, p-AKT(Ser473), mTOR, and ETV7 in control ( C ) and VD3-GNP-treated MDA-MB-231 cells. * p < 0.05. ( B ) Calculation of the protein expression (band density) in Western blot analysis (n = 3) in MDA-MB-231 cells. Protein levels of PI3K, AKT, p-AKT (Ser473), mTOR, and ETV7 were normalized by β-actin, * p < 0.05, ** p < 0.001. ( C ) representative Western blots of PI3K, AKT, p-AKT (Ser473), mTOR, and ETV7 in control ( C ), and VD3-GNP-treated MCF-7 cells. β-actin was used as a loading control. ( D ) The protein expression (band density) in Western blot analysis (n = 3) for protein levels in MCF-7 cells was normalized by β-actin, * p < 0.05, ** p < 0.001.

Article Snippet: PVDF membranes were blocked with 5% BSA in TBS-T at room temperature for 1 h and were subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with anti-mouse IgG (1:10,000, Jackson-Immuno Research, West Grove, PA, USA, 115035068) in TBS-T for 1 h. Primary antibodies for PI3K (Developmental Studies Hybridoma Bank (DSHB), AFFN-PIK3R2-3D4), mTOR (DSHB, CPTC-MTOR-1), AKT (DSHB, CPTC-AKT1-1), ETV7 (DSHB, PCRP-ETV7-1A1), YAP (DSHB, YAP2), Taz (DSHB, PCRP-ZBTB18-1B2), p-AKT (Ser473), and p-YAP (Ser127), (Cell Signaling Technology, Danvers, MA, USA, 9271) were used (1:100 in 0.5% TBS-T) and β-actin antibody conjugated to peroxidase was used as a loading control (Sigma-Aldrich, A3854, dilution 1:50,000).

Techniques: Western Blot, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Mitigation of Breast Cancer Cells’ Invasiveness via Down Regulation of ETV7, Hippo, and PI3K/mTOR Pathways by Vitamin D3 Gold-Nanoparticles

doi: 10.3390/ijms25105348

Figure Lengend Snippet: PPIs (using the STRING database) for the PI3K/mTOR/AKT (Gene IDs: PIK3C, MTOR, AKT1, ETV7) and Hippo pathways (Gene IDs: Yap1, TAZ), with 10 functional proteins. ( A ) AKT1; ( B ) MTOR; ( C ) PI3K; ( D ) ETV7; ( E ) YAP; ( F ) TAZ.

Article Snippet: PVDF membranes were blocked with 5% BSA in TBS-T at room temperature for 1 h and were subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with anti-mouse IgG (1:10,000, Jackson-Immuno Research, West Grove, PA, USA, 115035068) in TBS-T for 1 h. Primary antibodies for PI3K (Developmental Studies Hybridoma Bank (DSHB), AFFN-PIK3R2-3D4), mTOR (DSHB, CPTC-MTOR-1), AKT (DSHB, CPTC-AKT1-1), ETV7 (DSHB, PCRP-ETV7-1A1), YAP (DSHB, YAP2), Taz (DSHB, PCRP-ZBTB18-1B2), p-AKT (Ser473), and p-YAP (Ser127), (Cell Signaling Technology, Danvers, MA, USA, 9271) were used (1:100 in 0.5% TBS-T) and β-actin antibody conjugated to peroxidase was used as a loading control (Sigma-Aldrich, A3854, dilution 1:50,000).

Techniques: Functional Assay

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

Journal: International Journal of Molecular Sciences

Article Title: Mitigation of Breast Cancer Cells’ Invasiveness via Down Regulation of ETV7, Hippo, and PI3K/mTOR Pathways by Vitamin D3 Gold-Nanoparticles

doi: 10.3390/ijms25105348

Figure Lengend Snippet: Schematic of the PI3K/AKT/mTOR and Hippo pathways and their roles in various cellular mechanisms (growth, proliferation, etc.).

Article Snippet: PVDF membranes were blocked with 5% BSA in TBS-T at room temperature for 1 h and were subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with anti-mouse IgG (1:10,000, Jackson-Immuno Research, West Grove, PA, USA, 115035068) in TBS-T for 1 h. Primary antibodies for PI3K (Developmental Studies Hybridoma Bank (DSHB), AFFN-PIK3R2-3D4), mTOR (DSHB, CPTC-MTOR-1), AKT (DSHB, CPTC-AKT1-1), ETV7 (DSHB, PCRP-ETV7-1A1), YAP (DSHB, YAP2), Taz (DSHB, PCRP-ZBTB18-1B2), p-AKT (Ser473), and p-YAP (Ser127), (Cell Signaling Technology, Danvers, MA, USA, 9271) were used (1:100 in 0.5% TBS-T) and β-actin antibody conjugated to peroxidase was used as a loading control (Sigma-Aldrich, A3854, dilution 1:50,000).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Mitigation of Breast Cancer Cells’ Invasiveness via Down Regulation of ETV7, Hippo, and PI3K/mTOR Pathways by Vitamin D3 Gold-Nanoparticles

doi: 10.3390/ijms25105348

Figure Lengend Snippet: ( A ) Representative Western blots of PI3K, AKT, p-AKT(Ser473), mTOR, and ETV7 in control ( C ) and VD3-GNP-treated MDA-MB-231 cells. * p < 0.05. ( B ) Calculation of the protein expression (band density) in Western blot analysis (n = 3) in MDA-MB-231 cells. Protein levels of PI3K, AKT, p-AKT (Ser473), mTOR, and ETV7 were normalized by β-actin, * p < 0.05, ** p < 0.001. ( C ) representative Western blots of PI3K, AKT, p-AKT (Ser473), mTOR, and ETV7 in control ( C ), and VD3-GNP-treated MCF-7 cells. β-actin was used as a loading control. ( D ) The protein expression (band density) in Western blot analysis (n = 3) for protein levels in MCF-7 cells was normalized by β-actin, * p < 0.05, ** p < 0.001.

Article Snippet: PVDF membranes were blocked with 5% BSA in TBS-T at room temperature for 1 h and were subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with anti-mouse IgG (1:10,000, Jackson-Immuno Research, West Grove, PA, USA, 115035068) in TBS-T for 1 h. Primary antibodies for PI3K (Developmental Studies Hybridoma Bank (DSHB), AFFN-PIK3R2-3D4), mTOR (DSHB, CPTC-MTOR-1), AKT (DSHB, CPTC-AKT1-1), ETV7 (DSHB, PCRP-ETV7-1A1), YAP (DSHB, YAP2), Taz (DSHB, PCRP-ZBTB18-1B2), p-AKT (Ser473), and p-YAP (Ser127), (Cell Signaling Technology, Danvers, MA, USA, 9271) were used (1:100 in 0.5% TBS-T) and β-actin antibody conjugated to peroxidase was used as a loading control (Sigma-Aldrich, A3854, dilution 1:50,000).

Techniques: Western Blot, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Mitigation of Breast Cancer Cells’ Invasiveness via Down Regulation of ETV7, Hippo, and PI3K/mTOR Pathways by Vitamin D3 Gold-Nanoparticles

doi: 10.3390/ijms25105348

Figure Lengend Snippet: PPIs (using the STRING database) for the PI3K/mTOR/AKT (Gene IDs: PIK3C, MTOR, AKT1, ETV7) and Hippo pathways (Gene IDs: Yap1, TAZ), with 10 functional proteins. ( A ) AKT1; ( B ) MTOR; ( C ) PI3K; ( D ) ETV7; ( E ) YAP; ( F ) TAZ.

Article Snippet: PVDF membranes were blocked with 5% BSA in TBS-T at room temperature for 1 h and were subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with anti-mouse IgG (1:10,000, Jackson-Immuno Research, West Grove, PA, USA, 115035068) in TBS-T for 1 h. Primary antibodies for PI3K (Developmental Studies Hybridoma Bank (DSHB), AFFN-PIK3R2-3D4), mTOR (DSHB, CPTC-MTOR-1), AKT (DSHB, CPTC-AKT1-1), ETV7 (DSHB, PCRP-ETV7-1A1), YAP (DSHB, YAP2), Taz (DSHB, PCRP-ZBTB18-1B2), p-AKT (Ser473), and p-YAP (Ser127), (Cell Signaling Technology, Danvers, MA, USA, 9271) were used (1:100 in 0.5% TBS-T) and β-actin antibody conjugated to peroxidase was used as a loading control (Sigma-Aldrich, A3854, dilution 1:50,000).

Techniques: Functional Assay