Journal: Biology Open

Article Title: Generation and characterization of a knockout mouse of an enhancer of EBF3

doi: 10.1242/bio.062070

Figure Lengend Snippet: Ebf3 regulatory landscape and associated hs737/Rr169617 enhancer deletion mouse lines. Genome browser view of the topologically associating domain region containing Rr169617 and its target gene Ebf3 (GRCm38/mm10). The first track shows the two independent founder mouse lines generated in this study: line 299 (C57BL/6J-Rr169617 em1Tnt /J) and line 304 (C57BL/6J-Rr169617 em2Tnt /J). The second track shows the location of the regulatory region, Rr169617. The third track shows the location of human VISTA enhancers lifted over to the mouse genome. Included is hs737 that resides within the Rr169617 region. The fourth track shows enhancer-promoter interactions of Rr169617 and Ebf3 from . The fifth track shows the genes within the region. The sixth track shows human topologically associating domains lifted over to this region and show high conservation. Finally, the chromatin state data available from ENCODE3 is shown across the different timepoints in mouse development. The colors in this track refer to their chromHMM status as described in .

Article Snippet: Taqman mouse Ebf3 (Mm00438642_m1) and GAPDH (Mm99999915_g1) gene expression assays were performed on a QuantStudio 6 Flex quantitative thermocycler using four reactions for each sample.

Techniques: Generated

Journal: Biology Open

Article Title: Generation and characterization of a knockout mouse of an enhancer of EBF3

doi: 10.1242/bio.062070

Figure Lengend Snippet: qRT-PCR analysis for Ebf3 expression in E12.5 forebrain. (A) Results of five independent qRT-PCR for Ebf3 expression. Samples for each genotype were as follows: Rr169617 +/+ ( n =12), Rr169617 +/− ( n =14), and Rr169617 −/− ( n =10). (B) Relative fold expression aggregating data across all five independent qPCR experiments. For both A and B, relative fold expression is in comparison to the Rr169617 +/+ results.

Article Snippet: Taqman mouse Ebf3 (Mm00438642_m1) and GAPDH (Mm99999915_g1) gene expression assays were performed on a QuantStudio 6 Flex quantitative thermocycler using four reactions for each sample.

Techniques: Quantitative RT-PCR, Expressing, Comparison

Journal: Scientific Reports

Article Title: Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

doi: 10.1038/s41598-017-16685-8

Figure Lengend Snippet: Validation of VMP- and SU-specific transcript expression in female and male P0 rat tissues. Quantitative real-time PCR (qPCR) showed significantly elevated levels of both control (Fgf10, Ptn and Scube1) and candidate (Ebf3, Gfra3, Nmur2, Rspo2, Scara5, Slc26a7, Robo1 and Meis2) VMP-specific transcripts versus SU. Fgf10, Ptn, Scube1, Ebf3, Gfra3, Scara5 and Meis2 were expressed in VP and DP, while Rspo2, Nmur2 and Slc26a7 showed low expression in VP and DP. SU candidate transcripts Anxa1, Enpp2 and Unc5b were enriched versus VMP. Data is represented as mean fold difference to VMP ± SD of duplicate biological replicates and duplicate technical replicates (n = 4). Significance was detected using One-way ANOVA with TUKEY multiple comparison *p < 0.05. Figures in red indicate fold difference compared to VMP.

Article Snippet: Immunostaining of Ebf3 and Meis2 on serial sections of female and male rat P0 urogenital sinus tissue (isolated as per ) was performed as per using Ebf3 IgG (Clone 8D6, mouse monoclonal, Novus Biologicals, Littleton, Colorado, USA; dilution 1:1000) and Meis2 IgG (Clone 63-T, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, USA; dilution 1:750).

Techniques: Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Control, Comparison

Journal: Scientific Reports

Article Title: Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

doi: 10.1038/s41598-017-16685-8

Figure Lengend Snippet: Immunohistochemistry of Ebf3 and Meis2 in female VMP and male prostate. To examine the distribution of Ebf3 and Meis2 in mesenchymal subsets in VMP and ventral prostate (VP) we performed immunohistochemistry using P0 rat female and male reproductive tracts. Panels ( a–d ) show Ebf3 expression; in female ( a and c ) and male ( b and d ). Ebf3 showed a highly selective distribution within VMP and VP mesenchyme, and was absent from smooth muscle (SM) and the urethra ( c , a and b ). Panels ( e–h ) show Meis2 distribution; in female ( e and g ) and male ( f and h ) mesenchyme. Urethral epithelia (URE) and prostatic epithelia (E) were negative for both Ebf3 and Meis2. Scale bars ( a , b , e and f ) = 600 µm. Scale bars ( c , d , g and h ) = 300 µm.

Article Snippet: Immunostaining of Ebf3 and Meis2 on serial sections of female and male rat P0 urogenital sinus tissue (isolated as per ) was performed as per using Ebf3 IgG (Clone 8D6, mouse monoclonal, Novus Biologicals, Littleton, Colorado, USA; dilution 1:1000) and Meis2 IgG (Clone 63-T, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, USA; dilution 1:750).

Techniques: Immunohistochemistry, Expressing

Journal: Scientific Reports

Article Title: Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

doi: 10.1038/s41598-017-16685-8

Figure Lengend Snippet: Summary of primers used for qPCR.

Article Snippet: Immunostaining of Ebf3 and Meis2 on serial sections of female and male rat P0 urogenital sinus tissue (isolated as per ) was performed as per using Ebf3 IgG (Clone 8D6, mouse monoclonal, Novus Biologicals, Littleton, Colorado, USA; dilution 1:1000) and Meis2 IgG (Clone 63-T, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, USA; dilution 1:750).

Techniques:

Journal: Cancer Research

Article Title: Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

doi: 10.1158/0008-5472.can-09-3073

Figure Lengend Snippet: Figure 1. Interaction between frequently methylated genes and TGF-β pathway components in HNSCC. A, immunoprecipitation was done with SMAD2 antibody using lysates from WT SCC22B and SCC22B + IRX1 overexpression. This revealed no IRX1 association with SMAD2. B, chromatin immunoprecipitation analysis using EBF3 and SMAD2 antibodies in SCC22B cells. p21 and IRX1 promoter pulldown was assessed through quantitative PCR, which revealed increased pulldown of both promoters with ther respective antibody compared with the negative IgG control. C, immunoprecipitation analysis of SLC5A8 pulldown by survivin antibody. Arrow, SLC5A8, which is only pulled down by survivin and not by the IgG control. D, overexpression of SEPT9 in SCC22B and immunoprecipitation analysis of SEPT9 pulldown by HIF-1α antibody. HIF-1α associates with SEPT9, and this binding is increased in SEPT9-overexpressing cells (+SEPT9). Furthermore, SEPT9 expression correlates with decreased HIF-1α expression.

Article Snippet: The antibodies used in the study were as follows: IL-6 (Abcam, ab6672), ARTS (Sigma-Aldrich, A4471), SLC5A8 (Santa Cruz, sc-34189), SEPT9 (aka MSF; Abgent, AP6215a), EBF3 (Aviva Systems Biology, ARP31837_T100), survivin (Santa Cruz, sc-17779), α-tubulin (Cell Signaling Technologies), TGF-β1 (Santa Cruz, sc-146), caspase-3 (Cell Signaling, 9662), actin (Santa Cruz, sc-8432), HIF-1α (Cell Signaling, 3716), and involucrin (Aviva Systems Biology, ARP48675_P050).

Techniques: Methylation, Immunoprecipitation, Over Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Control, Binding Assay, Expressing

Journal: Cancer Research

Article Title: Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

doi: 10.1158/0008-5472.can-09-3073

Figure Lengend Snippet: Figure 3. EBF3 regulates p21 gene expression. A, luciferase analysis of p21 promoter activity in SCC22B cells with and without EBF3 overexpression. Overexpression of EBF3 significantly increases p21 promoter activity. *, P < 0.0006. B, quantitative RT-PCR analysis of p21 mRNA expression in SCC22B cells with and without EBF3 overexpression. EBF3 overexpression results in a significant increase in p21 transcription. *, P < 0.05.

Article Snippet: The antibodies used in the study were as follows: IL-6 (Abcam, ab6672), ARTS (Sigma-Aldrich, A4471), SLC5A8 (Santa Cruz, sc-34189), SEPT9 (aka MSF; Abgent, AP6215a), EBF3 (Aviva Systems Biology, ARP31837_T100), survivin (Santa Cruz, sc-17779), α-tubulin (Cell Signaling Technologies), TGF-β1 (Santa Cruz, sc-146), caspase-3 (Cell Signaling, 9662), actin (Santa Cruz, sc-8432), HIF-1α (Cell Signaling, 3716), and involucrin (Aviva Systems Biology, ARP48675_P050).

Techniques: Gene Expression, Luciferase, Activity Assay, Over Expression, Quantitative RT-PCR, Expressing

Journal: Cancer Research

Article Title: Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

doi: 10.1158/0008-5472.can-09-3073

Figure Lengend Snippet: Figure 4. Functional relevance of the interactions between the methylated candidates and components of the TGF-β pathway. A, Western blot analysis of ARTS, caspase-3, TGF-β, and involucrin expression in the five overexpressing cell lines (tubulin serves as a loading control). B, cell cycle analysis of the five overexpressing cell lines revealed a significant decrease in mitosis in IRX1-, EBF3-, and SLC5A8-overexpressing cells. *, P < 0.05; **, P < 0.01.

Article Snippet: The antibodies used in the study were as follows: IL-6 (Abcam, ab6672), ARTS (Sigma-Aldrich, A4471), SLC5A8 (Santa Cruz, sc-34189), SEPT9 (aka MSF; Abgent, AP6215a), EBF3 (Aviva Systems Biology, ARP31837_T100), survivin (Santa Cruz, sc-17779), α-tubulin (Cell Signaling Technologies), TGF-β1 (Santa Cruz, sc-146), caspase-3 (Cell Signaling, 9662), actin (Santa Cruz, sc-8432), HIF-1α (Cell Signaling, 3716), and involucrin (Aviva Systems Biology, ARP48675_P050).

Techniques: Functional Assay, Methylation, Western Blot, Expressing, Control, Cell Cycle Assay