Journal: Nucleic Acids Research

Article Title: Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition

doi: 10.1093/nar/gkw126

Figure Lengend Snippet: Integrative analysis identified ETS2, HNF4A, JUNB and FOXP1 as the putative master TFs of EMT. ( A ) Enrichment P -values of TF binding motifs in the proximal enhancers of partial-EMT high genes (x-axis) were plotted against the fold changes in gene expression during EMT (y-axis; 24 h versus 0 h, left panel; 72 h versus 0 h, right panel). Each circle represents a TF. The dotted lines represent the cutoff for the enrichment ( P -value < 0.05) and the expression change (absolute fold change ≥ 4). ( B ) Levels of ETS2, HNF4A, JUNB and FOXP1 mRNA during EMT as estimated using RNA-seq. ( C ) Immunoblots showing the levels of ETS2, HNF4A, JUNB and FOXP1 proteins during EMT. ( D ) Venn diagram showing the overlap of the targets of the five EMT TFs derived from published ChIP-seq data.

Article Snippet: 24 h into TGF-β-induced EMT, cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and probed with specific primary antibodies for ETS2 (Santa Cruz, sc-365666), HNF4A (Santa Cruz, sc-6557), JUNB (CST, 3753) or SMAD3 (CST, C67H9).

Techniques: Binding Assay, Gene Expression, Expressing, RNA Sequencing, Western Blot, Derivative Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition

doi: 10.1093/nar/gkw126

Figure Lengend Snippet: ETS2, HNF4A and JUNB are key regulators in a dynamic EMT Gene Regulatory Network model. ( A ) DREM output showing a dynamic gene regulatory map of EMT. The y-axis shows the levels of gene expression normalized to those at 0 h. Each line represents a path (cluster) of genes with a similar expression pattern, and nodes represent the hidden states of a hidden Markov model. The green nodes represent splitting points. For each splitting point, TFs with a split score < 0.001 were listed in ranked order of scores and were colored according to the expression level changes (blue for upregulation; red for downregulation). ( B ) Diagram illustrating the putative core regulatory network derived from DREM analysis. ( C ) Genome browser representation of ETS2, HNF4A, JUNB and FOXP1 binding events near ETS2 gene. ( D ) Same as (C) for HNF4A gene. ( E ) Same as (C) for JUNB gene.

Article Snippet: 24 h into TGF-β-induced EMT, cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and probed with specific primary antibodies for ETS2 (Santa Cruz, sc-365666), HNF4A (Santa Cruz, sc-6557), JUNB (CST, 3753) or SMAD3 (CST, C67H9).

Techniques: Gene Expression, Expressing, Derivative Assay, Binding Assay

Journal: Nucleic Acids Research

Article Title: Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition

doi: 10.1093/nar/gkw126

Figure Lengend Snippet: ETS2, HNF4A and JUNB play critical roles in partial-EMT. ( A ) Quantitative reverse transcription polymerase chain reaction results showing the levels of gene expression in A549 cells during TGF-β-induced EMT after silencing ETS2 expression using a specific siRNA. n = 3; error bars indicate mean ± SD. * P < 0.05; ** P < 0.01, determined using the two-tailed Student's t -test. ( B ) Same as (C) for silencing HNF4A. ( C ) Same as (A) for silencing JUNB. ( D ) Immunoblotting analysis of the protein abundance of indicated genes in A549 cells undergoing EMT treated with siRNAs targeting ETS2, HNF4A or JUNB. ( E ) A549 cells undergoing TGF-β-induced EMT were treated with siRNAs targeting ETS2, HNF4A or JUNB and were subjected to a migration assay. The migratory cells were quantified (bar charts). Scale bars: 100 μm. n = 6; error bars indicate mean ± SD. * P < 0.05; ** P < 0.01, determined using the two-tailed Student's t -test. ( F ) Same as (E) for the invasion assay. ( G ) Kaplan–Meier survival analysis based on the ETS2, HNF4A and JUNB expression levels in three independent Lung Adenocarcinoma data sets for disease-free survival.

Article Snippet: 24 h into TGF-β-induced EMT, cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and probed with specific primary antibodies for ETS2 (Santa Cruz, sc-365666), HNF4A (Santa Cruz, sc-6557), JUNB (CST, 3753) or SMAD3 (CST, C67H9).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Expressing, Two Tailed Test, Western Blot, Quantitative Proteomics, Migration, Invasion Assay

Journal: Nucleic Acids Research

Article Title: Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition

doi: 10.1093/nar/gkw126

Figure Lengend Snippet: ETS2, HNF4A and JUNB are synergistic master regulators of EMT. ( A ) Heatmap showing the colocalization of ETS2, HNF4A and JUNB at EMT-associated enhancers. The presence of ETS2, HNF4A and JUNB ChIP-seq peaks are displayed within a 6-kb window centered on the ETS2-bound site. ( B ) Summary plot for the ETS2, HNF4A and JUNB ChIP-seq peak enrichment across the ETS2-binding site associated with EMT genes. ( C ) Endogenous association of ETS2, HNF4A and JUNB. At 24 h into TGF-β-induced EMT, A549 cell lysates were prepared and were immunoprecipitated using the indicated antibody. The presence of associated proteins was then analyzed using immunoblotting. ( D ) Immunofluorescence staining for ETS2, HNF4A or JUNB in A549 cells stimulated with TGF-β for 24 h.

Article Snippet: 24 h into TGF-β-induced EMT, cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and probed with specific primary antibodies for ETS2 (Santa Cruz, sc-365666), HNF4A (Santa Cruz, sc-6557), JUNB (CST, 3753) or SMAD3 (CST, C67H9).

Techniques: ChIP-sequencing, Binding Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Staining

Journal: Nucleic Acids Research

Article Title: Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition

doi: 10.1093/nar/gkw126

Figure Lengend Snippet: Super-enhancers are associated with master EMT regulators. ( A ) The distribution of A549 H3K27ac tag intensities revealed 1050 super-enhancers. The red circles indicate EMT genes associated with super-enhancers. ( B ) Genome browser tracks showing the super-enhancers associated with ETS2, HNF4A, JUNB and SMAD3. The black bars denote super-enhancers. ( C ) Genome browser tracks showing super-enhancers associated with the EGFR gene and the binding events of ETS2, HNF4A and JUNB around EGFR gene. ( D ) Table depicting the enrichment of ETS2, HNF4a and JUNB binding motifs in EMT-associated super-enhancers relative to the genomic background. ( E ) Bar plots showing the enriched association of EMT genes with super-enhancers. Bars represent the observed number of super-enhancer-associated EMT genes and circles represent the expected number of super-enhancer-associated genes assuming no enrichment. * P < 0.001, determined using Fisher's Exact Test. ( F ) Bar plots showing the universal association of EMT genes with active enhancers. Bars represent the observed percentages of active-enhancer-associated EMT genes.

Article Snippet: 24 h into TGF-β-induced EMT, cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and probed with specific primary antibodies for ETS2 (Santa Cruz, sc-365666), HNF4A (Santa Cruz, sc-6557), JUNB (CST, 3753) or SMAD3 (CST, C67H9).

Techniques: Binding Assay

Journal: Aging Cell

Article Title: Linkage of Cardiac Gene Expression Profiles and ETS2 with Lifespan Variability in Rats

doi: 10.1111/j.1474-9726.2012.00794.x

Figure Lengend Snippet: (A) Immunoblot and data analysis for ETS1 and ETS2 in F344 rat heart from each relative mortality group (4, 8, 30 and 80%). (B) ETS2 immunostaining of heart tissues from F/N rats at a) 2, b) 16, c) 24 and d) 32 months of age. ETS2 (Brown) and TUNEL (Violet) co-staining of hearts from 16 month old animals are shown in e–g, with magnifications shown in f and h. Dark brown staining in e illustrates high levels of ETS2 shown in selected regions of myocardium. Arrows indicate TUNEL positive CMs (f). g and h show TUNEL positive non-CMs lacking strong ETS2 staining. (C) Immunoblot and data analysis for ETS1 and ETS2 protein abundance in rFB, NNCM, and adult left ventricular cardiomyocytes (AdCM), p<0.05. (D) Immunoblot and data analysis for ETS2 protein abundance in vehicle-treated NNCM (Ctls) and NNCMs treated for 24 hours with 5μM of ISO, PE and AngII, * p<0.01.

Article Snippet: In performing these studies, the ETS2 antibody (sc351, Santa-Cruz) proved highly specific for its intended target; however, two different Ets1 antibodies (sc350 and sc111, Santa-Cruz) demonstrated weak cross-reactivity with ETS2. (B).

Techniques: Western Blot, Immunostaining, TUNEL Assay, Staining, Quantitative Proteomics

Journal: Aging Cell

Article Title: Linkage of Cardiac Gene Expression Profiles and ETS2 with Lifespan Variability in Rats

doi: 10.1111/j.1474-9726.2012.00794.x

Figure Lengend Snippet: (A) Adenovirus-mediated overexpression of ETS1 and ETS2 in CMs. In performing these studies, the ETS2 antibody (sc351, Santa-Cruz) proved highly specific for its intended target; however, two different Ets1 antibodies (sc350 and sc111, Santa-Cruz) demonstrated weak cross-reactivity with ETS2. (B). Images showing a) GFP expression, b) ETS2 immunostaining restricted primarily to the nucleus, c) DAPI nuclear stain and d) a composite image. (C) NNCMs were are infected with Ad-Ets2 for 24 hours. Images show a) ETS2 immunostaining, b) Troponin-T immunostaining, c) DAPI staining and d) a composite image. Arrows indicated condensed nuclei. Size marker=100μm. (D) ETS2 over-expression selectively promotes large-scale DNA in fragmentation in NNCMs 24 hours after infection. At 48 hours, internucleosomal fragmentation was observed. ETS2-mediated high molecular weight and internucleosomal fragementation could not be inhibited by pretreatment with 60μM Z-VAD-FMK; however, caspase-dependent apoptosis could be induced with 0.5 mM Staurosporine, which could be inhibited by pre-treatment with 60 μM Z- VAD-FMK. (E) Pro-caspase 3, but not cleaved Caspase 3 (175Asp) was detected in NNCMs infected with Ad-ETS1 or Ad-ETS2; however, infected rFBs contained elevated levels of cleaved Caspase3. (F) In rFBs, ETS2 over-expression, but not GFP, promotes DNA fragmentation that can be inhibited by 50μM Z-VAD-FMK.

Article Snippet: In performing these studies, the ETS2 antibody (sc351, Santa-Cruz) proved highly specific for its intended target; however, two different Ets1 antibodies (sc350 and sc111, Santa-Cruz) demonstrated weak cross-reactivity with ETS2. (B).

Techniques: Over Expression, Expressing, Immunostaining, Staining, Infection, Marker, High Molecular Weight

Journal: The Journal of biological chemistry

Article Title: Oncostatin M stimulates c-Fos to bind a transcriptionally responsive AP-1 element within the tissue inhibitor of metalloproteinase-1 promoter.

doi: 10.1074/jbc.273.9.5211

Figure Lengend Snippet: FIG. 7. SP1 nuclear factor binding to the TIMP-1 promoter. A, an excess of cold specific unlabeled competitor oligonucleotides (cold SP1) or irrelevant competitor (cold AP-1/Ets, probe 3) were used to identify specific binding of SP1 (probe 6) gel-shifted complexes. Free probe migrates at the bottom of the gel. B, nuclear extracts from cells stimulated with OSM for 0, 0.25, 1, or 12 h were incubated with a TIMP-1 putative SP1 DNA binding element (probe 6) binding examined by EMSA analysis. C, anti-SP1 antibody was used to supershift SP1 binding to probe 6. Supershifts were inhibited by preincubation of anti-SP1 antibody with SP1 peptide but not by an irrelevant peptide (Fos peptide). The open circle (E) represents the position of nonspecific complexes.

Article Snippet: Rabbit polyclonal antibodies against Fos/Jun (anti-pan-Fos, anti-c-Fos, anti-c-Jun, anti-JunB, anti-JunD), SP1, STAT3 and Ets-1/Ets-2 nuclear factors, and specific peptides were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Binding Assay, Incubation