anti-cdx2 Search Results


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  • 93
    Millipore anti cdx2
    Effect of CSF2 and sex on cleavage and development of embryos to the blastocyst stage. The top panel shows representative images of a female (A) and a male (B) Day 7 blastocyst labeled with Hoechst (all nuclei) and <t>anti-CDX2</t> (trophectoderm (TE) cells). The graphs represent least-squares means ± s.e.m. for cleavage (C), percent putative zygotes developing to the blastocyst stage (D), numbers of total (E), inner cell mass (ICM; F), trophectoderm (TE; G), and the ratio of TE to ICM cell numbers (H). The experiment was replicated nine times with a total of 351–427 putative zygotes per treatment group. P value for the main effects of sex, CSF2 treatment (trt) and the interaction are shown for all effects where P
    Anti Cdx2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti cdx2
    Identification of lung nodules to be of colon origin using <t>CDX2</t> as a colon specific biomarker
    Anti Cdx2, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cdx2/product/Abcam
    Average 95 stars, based on 92 article reviews
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    95
    Abcam rabbit anti cdx2
    Elevated OCT3/4 levels and earlier restriction of OCT3/4 to the inner cells in p66Shc knockdown blastocysts. Embryos were categorized according to cell number before analysis: 32–64 cells (scrambled mean ± SD = 51.94 ± 8.33, p66Shc KD mean = 49.60 ± 7.93) or 64–128 cells (scrambled mean = 71.38 ± 5.48, p66Shc KD mean = 74.76 ± 4.46). (A) Representative confocal microscopy images of scrambled and p66Shc knockdown 32–64-cell blastocysts immunostained for <t>CDX2</t> and OCT3/4. Scale bars are 20 μm. (B) Representative confocal microscopy images of scrambled and p66Shc knockdown 64–128-cell blastocysts. Scale bars are 20 μm. (C) p66Shc knockdown blastocysts have similar CDX2-positive/OCT3/4-positive nuclei compared with scrambled controls in 32–64-cell blastocysts ( n = 19, mean ± SEM, Students' t -test). (D) p66Shc knockdown blastocysts have significantly fewer CDX2-positive/OCT3/4-positive nuclei in 64–128-cell blastocyst ( n = 23, * P
    Rabbit Anti Cdx2, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti cdx2/product/Abcam
    Average 95 stars, based on 96 article reviews
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    90
    Biogenex anti cdx2
    Confluence-dependent upregulation of transcription factors in HCT-116 and HCT-15 cells . Culture conditions were the same as for Figure 1. Protein expression was analyzed by Western blotting as described in Materials and Methods. Data shown are representative of three independent experiments. All transcription factors exhibited altered protein expression levels in a confluence-dependent manner except for HNF-1α, which was unchanged. * N/D , <t>Cdx2</t> was not detected in HCT-116.
    Anti Cdx2, supplied by Biogenex, used in various techniques. Bioz Stars score: 90/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson anti cdx2
    Model for dynamic <t>CDX2</t> function during intestinal differentiation In proliferating cells, CDX2 binds with relative selectivity, frequently accompanied by GATA6, at H3K4Me2 regions with selectively open chromatin at loci active in progenitor cells. Genes associated with epithelial maturity are not expressed in these cells and their enhancers have a relatively closed structure with less H3K4Me2. As cells differentiate, CDX2 relocates from progenitor-specific target genes to those associated with maturity, now accompanied by HNF4A; the appearance of an active H3K4Me2 pattern at many loci depends on CDX2.
    Anti Cdx2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Genex Corp anti cdx2
    Immunostaining of <t>CDX2.</t> (A) IVF-derived hatched blastocysts (100X) and (B) TE cells produced under feeder-free conditions showing positive expression of CDX2 (200X). Scale bar = 100 μm.
    Anti Cdx2, supplied by Genex Corp, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cdx2/product/Genex Corp
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    92
    Novocastra cdx2
    (A) Kaplan–Meier survival curve showing effect of <t>Cdx2</t> expression on overall survival ( n = 93). (B) ROC curve highlighting the strong predictive effect and specificity of loss of Cdx2 expression for MMR-deficiency. (C) Kaplan–Meier survival curve showing effect of Cdx2 expression on overall survival in MMR-proficient colorectal cancer patients only ( n = 78). Wilcoxon’s test.
    Cdx2, supplied by Novocastra, used in various techniques. Bioz Stars score: 92/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies cdx2
    (A) Kaplan–Meier survival curve showing effect of <t>Cdx2</t> expression on overall survival ( n = 93). (B) ROC curve highlighting the strong predictive effect and specificity of loss of Cdx2 expression for MMR-deficiency. (C) Kaplan–Meier survival curve showing effect of Cdx2 expression on overall survival in MMR-proficient colorectal cancer patients only ( n = 78). Wilcoxon’s test.
    Cdx2, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdx2/product/Agilent technologies
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    85
    Millipore anti cdx 2 mab
    (A) Kaplan–Meier survival curve showing effect of <t>Cdx2</t> expression on overall survival ( n = 93). (B) ROC curve highlighting the strong predictive effect and specificity of loss of Cdx2 expression for MMR-deficiency. (C) Kaplan–Meier survival curve showing effect of Cdx2 expression on overall survival in MMR-proficient colorectal cancer patients only ( n = 78). Wilcoxon’s test.
    Anti Cdx 2 Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology anti cdx2
    The mean apoptotic rate in <t>MGC-803/Cdx2</t> small interference RNA cells was significantly higher than that in MGC-803/Cdx2 negative control or MGC-803 cells. Percentages of apoptotic cells analyzed by flow cytometry. Numbers in the quadrants reflected the
    Anti Cdx2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Marque anti cdx2
    Upper panel: qPCR analysis of <t>CDX2-negative</t> HT29 cells treated with increasing concentrations of the DNMTi decitabine (1.25 μM, 2.5 μM, 5 μM, 10 μM) for 48 h and increasing concentrations of the HDAC4/5i LMK-235 (5 nM, 10 nM, 20 nM, 40 nM, 80 nM). Data were normalized to the HMBS housekeeping gene and are shown as n -fold regulation compared with DMSO-treated cells. MWU: *** p
    Anti Cdx2, supplied by Cell Marque, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Ventana Medical cdx2
    Immunohistochemical (immunoperoxidase and hematoxylin counterstain) findings of synchronous primary carcinoid tumor and primary adenocarcinoma arising within mature cystic teratoma of horseshoe kidney . (A) <t>CDX2</t> staining was positive in the mucinous columnar enteric-type or colonic-like epithelium lining of teratomatous cysts. Original magnification ×200. (B) Cytokeratin 20 staining was positive in the mucinous columnar enteric-type or colonic-like epithelium lining of teratomatous cysts. Original magnification ×200. (C) Smooth muscle actin was positive in teratomatous cyst wall. Original magnification ×200. (D) Cytokeratin 7 staining was positive in the adenocarcinoma component in right upper part of figure, but negative in the carcinoid tumor component in left lower part of figure. Original magnification ×200.
    Cdx2, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 93/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cdx2  (Bethyl)
    93
    Bethyl cdx2
    Depletion of architectural proteins alters histone modifications and transcription factor occupancy. ( A )–( C ) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t -test, P -values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. ( D )–( F ) ChIP for FOXA1 (D), FOXA2 (E) and <t>CDX2</t> (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. * P
    Cdx2, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Orbigen cdx2
    Depletion of architectural proteins alters histone modifications and transcription factor occupancy. ( A )–( C ) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t -test, P -values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. ( D )–( F ) ChIP for FOXA1 (D), FOXA2 (E) and <t>CDX2</t> (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. * P
    Cdx2, supplied by Orbigen, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of CSF2 and sex on cleavage and development of embryos to the blastocyst stage. The top panel shows representative images of a female (A) and a male (B) Day 7 blastocyst labeled with Hoechst (all nuclei) and anti-CDX2 (trophectoderm (TE) cells). The graphs represent least-squares means ± s.e.m. for cleavage (C), percent putative zygotes developing to the blastocyst stage (D), numbers of total (E), inner cell mass (ICM; F), trophectoderm (TE; G), and the ratio of TE to ICM cell numbers (H). The experiment was replicated nine times with a total of 351–427 putative zygotes per treatment group. P value for the main effects of sex, CSF2 treatment (trt) and the interaction are shown for all effects where P

    Journal: Reproduction (Cambridge, England)

    Article Title: Sex differences in response of the bovine embryo to colony-stimulating factor 2

    doi: 10.1530/REP-16-0336

    Figure Lengend Snippet: Effect of CSF2 and sex on cleavage and development of embryos to the blastocyst stage. The top panel shows representative images of a female (A) and a male (B) Day 7 blastocyst labeled with Hoechst (all nuclei) and anti-CDX2 (trophectoderm (TE) cells). The graphs represent least-squares means ± s.e.m. for cleavage (C), percent putative zygotes developing to the blastocyst stage (D), numbers of total (E), inner cell mass (ICM; F), trophectoderm (TE; G), and the ratio of TE to ICM cell numbers (H). The experiment was replicated nine times with a total of 351–427 putative zygotes per treatment group. P value for the main effects of sex, CSF2 treatment (trt) and the interaction are shown for all effects where P

    Article Snippet: As a negative control (two embryos per procedure), anti-CDX2 was replaced with 1 µg/mL mouse IgG (Sigma-Aldrich) diluted in antibody buffer (0.1% (v/v) Tween 20 and 1% (w/v) BSA in DPBS).

    Techniques: Labeling

    Immunohistochemical staining of the appendiceal and ovarian masses. The tumor cells of the right ovarian mass were (A) strongly positive for CK7, and negative for (B) CK20 and (C) CDX2. The tumor cells of the appendiceal mass were (D) negative for CK7 and positive for (E) CK20 and (F) CDX2. CK, cytokeratin; CDX2, homeobox protein CDX-2.

    Journal: Oncology Letters

    Article Title: An unusual case of pseudomyxoma peritonei associated with synchronous primary mucinous tumors of the ovary and appendix: A case report

    doi: 10.3892/ol.2017.6079

    Figure Lengend Snippet: Immunohistochemical staining of the appendiceal and ovarian masses. The tumor cells of the right ovarian mass were (A) strongly positive for CK7, and negative for (B) CK20 and (C) CDX2. The tumor cells of the appendiceal mass were (D) negative for CK7 and positive for (E) CK20 and (F) CDX2. CK, cytokeratin; CDX2, homeobox protein CDX-2.

    Article Snippet: Monoclonal CK7 (dilution, 1:100; cat. no., NCL-L-CK7-OVTL), CK20 (dilution, 1:100; cat. no., NCL-L-CK20-561; both from Novocastra; Leica Microsystems GmbH) and CDX2 antibodies (dilution 1:250; cat. no., 235R-15; Sigma Alrich; Merck KGaA) were applied on the slides.

    Techniques: Immunohistochemistry, Staining

    Identification of lung nodules to be of colon origin using CDX2 as a colon specific biomarker

    Journal: Experimental and toxicologic pathology : official journal of the Gesellschaft fur Toxikologische Pathologie

    Article Title: Characterization of azoxymethane-induced colon tumor metastasis to lung in a mouse model relevant to human sporadic colorectal cancer and evaluation of grape seed extract efficacy

    doi: 10.1016/j.etp.2014.02.003

    Figure Lengend Snippet: Identification of lung nodules to be of colon origin using CDX2 as a colon specific biomarker

    Article Snippet: Purchased antibodies include anti-CDX2, anti-CK20, anti-Surfactant D and anti-TTF-1 (all from Abcam).

    Techniques: Biomarker Assay

    Effects of bile acids on CDX2 and MUC2 expression in GES-1 cells. (A and B) GES-1 cells were treated with DMSO or various doses of bile acids (100, 200, 400 and 600 µ mol/l) for different periods of time (6, 12, 24 and 48 h). Cell viability was then determined using a Cell Counting Kit 8 assay. (C and D) GES-1 cells were treated with DMSO or various concentrations of bile acids (50, 100, 150 and 200 µ mol/l) for 24 h. Reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of CDX2 and MUC2 in GES-1 cells treated with bile acids. (E) Western blot analysis of CDX2 and MUC2 protein expression in each group of GES-1 cells stimulated with CDCA. (F) Comparison of CDX2 and MUC2 protein levels in each group of cells stimulated with CDCA. (G) Western blot analysis of CDX2 and MUC2 protein expression in each group of GES-1 cells stimulated with DCA. (H) Comparison of CDX2 and MUC2 protein levels in each group of cells stimulated with DCA. (I and J) GES-1 cells treated with various concentrations of bile acids were transfected with 2.5 kb CDX2- or 2.6 kb MUC2-luc promoter constructs. A dual-luciferase reporter assay was conducted to determine (I) CDX2 and (J) MUC2 promoter activity. Data are presented as the means ± standard deviation from three independent experiments. * P

    Journal: International Journal of Oncology

    Article Title: Bile acids promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/NF-κB signalling pathway

    doi: 10.3892/ijo.2019.4692

    Figure Lengend Snippet: Effects of bile acids on CDX2 and MUC2 expression in GES-1 cells. (A and B) GES-1 cells were treated with DMSO or various doses of bile acids (100, 200, 400 and 600 µ mol/l) for different periods of time (6, 12, 24 and 48 h). Cell viability was then determined using a Cell Counting Kit 8 assay. (C and D) GES-1 cells were treated with DMSO or various concentrations of bile acids (50, 100, 150 and 200 µ mol/l) for 24 h. Reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of CDX2 and MUC2 in GES-1 cells treated with bile acids. (E) Western blot analysis of CDX2 and MUC2 protein expression in each group of GES-1 cells stimulated with CDCA. (F) Comparison of CDX2 and MUC2 protein levels in each group of cells stimulated with CDCA. (G) Western blot analysis of CDX2 and MUC2 protein expression in each group of GES-1 cells stimulated with DCA. (H) Comparison of CDX2 and MUC2 protein levels in each group of cells stimulated with DCA. (I and J) GES-1 cells treated with various concentrations of bile acids were transfected with 2.5 kb CDX2- or 2.6 kb MUC2-luc promoter constructs. A dual-luciferase reporter assay was conducted to determine (I) CDX2 and (J) MUC2 promoter activity. Data are presented as the means ± standard deviation from three independent experiments. * P

    Article Snippet: Chromatin-protein complexes were immunoprecipitated with 5 µ g anti-CDX2, anti-p50 and anti-p65 antibodies, or 1 µ g rabbit immunoglobulin G (cat. no. ab171870; Abcam) as a negative control. qPCR was performed to amplify the regions of interest or internal negative control regions.

    Techniques: Expressing, Cell Counting, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Construct, Luciferase, Reporter Assay, Activity Assay, Standard Deviation

    FXR is involved in the regulation of bile acid-induced CDX2 and MUC2 expression. (A) Dual-luciferase reporter assay was conducted to determine the effect of knockdown of CDX2 expression on bile acid-enhanced MUC2 promoter activity. (B) Western blotting was conducted to determine the effect of knockdown of CDX2 on bile acid-enhanced MUC2 expression. (C) Comparison of CDX2 and MUC2 protein levels in each group of GES-1 cells. (D) Mutagenesis of the MUC2 promoter was performed using a site-directed mutagenesis kit. A dual-luciferase reporter assay was conducted to detect the luciferase activities in GES-1 cells transfected with a MUC2 promoter reporter construct or mutated construct. (E) A quantitative chromatin immunopre-cipitation assay was performed to investigate the effect of bile acids on the binding of CDX2 to the MUC2 promoter. (F) Reverse transcription-quantitative polymerase chain reaction was conducted to detect the mRNA expression levels of FXR in cells treated with DMSO, or CDCA or DCA at various concentrations (0, 100, 150 and 200 µ mol/l) for 24 h. (G) Western blot analysis of FXR protein expression in each group of bile acid-treated GES-1 cells. (H) Comparison of FXR protein levels in each group of GES-1 cells stimulated with CDCA or DCA. (I) siRNA-FXR was transfected into cells with the CDX2-luc promoter construct. A dual-luciferase reporter assay was conducted to determine the effect of the downregulation of FXR on bile acid-enhanced CDX2 promoter activity. (J) Effects of treatment with the FXR agonist GW4064 on bile acid-enhanced CDX2 promoter activity. (K) Western blotting was performed to investigate the effect of FXR downregulation on bile acid-induced CDX2 and MUC2 expression. (L) Comparison of CDX2, MUC2 and FXR protein expression in each group of GES-1 cells. Data are presented as the means ± standard deviation from three independent experiments. * P

    Journal: International Journal of Oncology

    Article Title: Bile acids promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/NF-κB signalling pathway

    doi: 10.3892/ijo.2019.4692

    Figure Lengend Snippet: FXR is involved in the regulation of bile acid-induced CDX2 and MUC2 expression. (A) Dual-luciferase reporter assay was conducted to determine the effect of knockdown of CDX2 expression on bile acid-enhanced MUC2 promoter activity. (B) Western blotting was conducted to determine the effect of knockdown of CDX2 on bile acid-enhanced MUC2 expression. (C) Comparison of CDX2 and MUC2 protein levels in each group of GES-1 cells. (D) Mutagenesis of the MUC2 promoter was performed using a site-directed mutagenesis kit. A dual-luciferase reporter assay was conducted to detect the luciferase activities in GES-1 cells transfected with a MUC2 promoter reporter construct or mutated construct. (E) A quantitative chromatin immunopre-cipitation assay was performed to investigate the effect of bile acids on the binding of CDX2 to the MUC2 promoter. (F) Reverse transcription-quantitative polymerase chain reaction was conducted to detect the mRNA expression levels of FXR in cells treated with DMSO, or CDCA or DCA at various concentrations (0, 100, 150 and 200 µ mol/l) for 24 h. (G) Western blot analysis of FXR protein expression in each group of bile acid-treated GES-1 cells. (H) Comparison of FXR protein levels in each group of GES-1 cells stimulated with CDCA or DCA. (I) siRNA-FXR was transfected into cells with the CDX2-luc promoter construct. A dual-luciferase reporter assay was conducted to determine the effect of the downregulation of FXR on bile acid-enhanced CDX2 promoter activity. (J) Effects of treatment with the FXR agonist GW4064 on bile acid-enhanced CDX2 promoter activity. (K) Western blotting was performed to investigate the effect of FXR downregulation on bile acid-induced CDX2 and MUC2 expression. (L) Comparison of CDX2, MUC2 and FXR protein expression in each group of GES-1 cells. Data are presented as the means ± standard deviation from three independent experiments. * P

    Article Snippet: Chromatin-protein complexes were immunoprecipitated with 5 µ g anti-CDX2, anti-p50 and anti-p65 antibodies, or 1 µ g rabbit immunoglobulin G (cat. no. ab171870; Abcam) as a negative control. qPCR was performed to amplify the regions of interest or internal negative control regions.

    Techniques: Expressing, Luciferase, Reporter Assay, Activity Assay, Western Blot, Mutagenesis, Transfection, Construct, Binding Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Bile acids induce molecular alterations linked to premalignant lesions in murine gastric mucosa in vivo . (A) Western blot analysis of MUC2, CDX2, p50, p65 and FXR protein expression in murine gastric mucosa samples from each group. Comparison of (B) MUC2, (C) CDX2, (D) p50, (E) p65 and (F) FXR protein expression levels in each group. * P

    Journal: International Journal of Oncology

    Article Title: Bile acids promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/NF-κB signalling pathway

    doi: 10.3892/ijo.2019.4692

    Figure Lengend Snippet: Bile acids induce molecular alterations linked to premalignant lesions in murine gastric mucosa in vivo . (A) Western blot analysis of MUC2, CDX2, p50, p65 and FXR protein expression in murine gastric mucosa samples from each group. Comparison of (B) MUC2, (C) CDX2, (D) p50, (E) p65 and (F) FXR protein expression levels in each group. * P

    Article Snippet: Chromatin-protein complexes were immunoprecipitated with 5 µ g anti-CDX2, anti-p50 and anti-p65 antibodies, or 1 µ g rabbit immunoglobulin G (cat. no. ab171870; Abcam) as a negative control. qPCR was performed to amplify the regions of interest or internal negative control regions.

    Techniques: In Vivo, Western Blot, Expressing

    BAs promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/nuclear factor-κB signalling pathway. BAs enter the cell by active transport or by diffusion and induce the upregulation and nuclear location of p50/p65 expression. Nuclear p50 binds to the CDX2 promoter and promotes CDX2 transactivation. The BA-induced increase in FXR expression enhances the binding of p50 to the CDX2 promoter. The CDX2 protein then binds to the MUC2 promoter and increases MUC2 transactivation. ASBT, apical sodium-dependent bile acid transporter; BA, bile acid; CDX2, caudal-related homeobox transcription factor 2; FXR, farnesoid X receptor; MUC2, mucin 2.

    Journal: International Journal of Oncology

    Article Title: Bile acids promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/NF-κB signalling pathway

    doi: 10.3892/ijo.2019.4692

    Figure Lengend Snippet: BAs promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/nuclear factor-κB signalling pathway. BAs enter the cell by active transport or by diffusion and induce the upregulation and nuclear location of p50/p65 expression. Nuclear p50 binds to the CDX2 promoter and promotes CDX2 transactivation. The BA-induced increase in FXR expression enhances the binding of p50 to the CDX2 promoter. The CDX2 protein then binds to the MUC2 promoter and increases MUC2 transactivation. ASBT, apical sodium-dependent bile acid transporter; BA, bile acid; CDX2, caudal-related homeobox transcription factor 2; FXR, farnesoid X receptor; MUC2, mucin 2.

    Article Snippet: Chromatin-protein complexes were immunoprecipitated with 5 µ g anti-CDX2, anti-p50 and anti-p65 antibodies, or 1 µ g rabbit immunoglobulin G (cat. no. ab171870; Abcam) as a negative control. qPCR was performed to amplify the regions of interest or internal negative control regions.

    Techniques: Expressing, Diffusion-based Assay, Binding Assay

    NF-κB is involved in the regulation of bile acid-induced FXR/CDX2/MUC2 signalling pathway activation. (A) Dual-luciferase reporter assay was conducted to determine the effect of bile acids on NF-κB activity. (B) Western blotting was conducted to detect the effects of bile acids on p50/p65 protein expression. (C) Comparison of p50/p65 protein levels in each group of GES-1 cells. (D) Dual-luciferase reporter assay was performed to detect the effect of the FXR agonist GW4064 or the antagonist Z-gug on bile acid-enhanced NF-κB activity. (E) Western blotting was conducted to determine the effect of the FXR agonist GW4064 or the antagonist Z-gug on bile acid-induced alterations in p50 and p65 protein levels. (F) Comparison of p50 and p65 protein levels in each group of GES-1 cells. (G) Dual-luciferase reporter assay was conducted to determine the effect of the NF-κB inhibitor PDTC on bile acid-enhanced CDX2 promoter activity. (H) Western blotting was performed to determine the effect of the NF-κB inhibitor PDTC on bile acid-induced alterations in CDX2 and MUC2 protein expression levels. (I) Comparison of CDX2 and MUC2 protein levels in each group of GES-1 cells. (J) Dual-luciferase reporter assay was conducted to detect the luciferase activities in GES-1 cells transfected with CDX2 promoter reporter construct or mutated construct. (K and L) Quantitative chromatin immunoprecipitation assay was performed to investigate the effect of the FXR agonist GW4064 or the antagonist Z-gug on the binding of NF-κB (K) p50 or (L) p65 protein to the CDX2 promoter. Data are presented as the means ± standard deviation from three independent experiments. * P

    Journal: International Journal of Oncology

    Article Title: Bile acids promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/NF-κB signalling pathway

    doi: 10.3892/ijo.2019.4692

    Figure Lengend Snippet: NF-κB is involved in the regulation of bile acid-induced FXR/CDX2/MUC2 signalling pathway activation. (A) Dual-luciferase reporter assay was conducted to determine the effect of bile acids on NF-κB activity. (B) Western blotting was conducted to detect the effects of bile acids on p50/p65 protein expression. (C) Comparison of p50/p65 protein levels in each group of GES-1 cells. (D) Dual-luciferase reporter assay was performed to detect the effect of the FXR agonist GW4064 or the antagonist Z-gug on bile acid-enhanced NF-κB activity. (E) Western blotting was conducted to determine the effect of the FXR agonist GW4064 or the antagonist Z-gug on bile acid-induced alterations in p50 and p65 protein levels. (F) Comparison of p50 and p65 protein levels in each group of GES-1 cells. (G) Dual-luciferase reporter assay was conducted to determine the effect of the NF-κB inhibitor PDTC on bile acid-enhanced CDX2 promoter activity. (H) Western blotting was performed to determine the effect of the NF-κB inhibitor PDTC on bile acid-induced alterations in CDX2 and MUC2 protein expression levels. (I) Comparison of CDX2 and MUC2 protein levels in each group of GES-1 cells. (J) Dual-luciferase reporter assay was conducted to detect the luciferase activities in GES-1 cells transfected with CDX2 promoter reporter construct or mutated construct. (K and L) Quantitative chromatin immunoprecipitation assay was performed to investigate the effect of the FXR agonist GW4064 or the antagonist Z-gug on the binding of NF-κB (K) p50 or (L) p65 protein to the CDX2 promoter. Data are presented as the means ± standard deviation from three independent experiments. * P

    Article Snippet: Chromatin-protein complexes were immunoprecipitated with 5 µ g anti-CDX2, anti-p50 and anti-p65 antibodies, or 1 µ g rabbit immunoglobulin G (cat. no. ab171870; Abcam) as a negative control. qPCR was performed to amplify the regions of interest or internal negative control regions.

    Techniques: Activation Assay, Luciferase, Reporter Assay, Activity Assay, Western Blot, Expressing, Transfection, Construct, Chromatin Immunoprecipitation, Binding Assay, Standard Deviation

    CDX2 expression is correlated with FXR and MUC2 expression in gastric IM tissues. (A) Immunohistochemical staining of CDX2, MUC2 and FXR in gastric mucosal tissues with or without IM. Scale bar, 100 µ m. (B) CDX2, (D) MUC2 and (F) FXR staining was classified into negative and positive, and the percentage of tissues in each group was shown. IRS of (C) CDX2, (E) MUC2 and (G) FXR staining in normal mucosa and IM tissues. Reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of (H) CDX2, (I) MUC2 and (J) FXR in mucosal tissues with or without IM. Correlations between the mRNA expression levels of CDX2 and (K) MUC2 (r=0.3602; P

    Journal: International Journal of Oncology

    Article Title: Bile acids promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/NF-κB signalling pathway

    doi: 10.3892/ijo.2019.4692

    Figure Lengend Snippet: CDX2 expression is correlated with FXR and MUC2 expression in gastric IM tissues. (A) Immunohistochemical staining of CDX2, MUC2 and FXR in gastric mucosal tissues with or without IM. Scale bar, 100 µ m. (B) CDX2, (D) MUC2 and (F) FXR staining was classified into negative and positive, and the percentage of tissues in each group was shown. IRS of (C) CDX2, (E) MUC2 and (G) FXR staining in normal mucosa and IM tissues. Reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of (H) CDX2, (I) MUC2 and (J) FXR in mucosal tissues with or without IM. Correlations between the mRNA expression levels of CDX2 and (K) MUC2 (r=0.3602; P

    Article Snippet: Chromatin-protein complexes were immunoprecipitated with 5 µ g anti-CDX2, anti-p50 and anti-p65 antibodies, or 1 µ g rabbit immunoglobulin G (cat. no. ab171870; Abcam) as a negative control. qPCR was performed to amplify the regions of interest or internal negative control regions.

    Techniques: Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

    Colonic lineage specificity and potency to produce phenotypical diversity in OSK-V50 cells. (A) Immunocytochemistry for CDX2 and CK20 protein. OSK-V50 cells were positive for CDX2 and CK20, as well as M-V0 cells. The cells were counterstained with Hoechst33342. Scale bars: 100 µm. (B) The phenotypical diversity in morphology and mobility of derivatives of OSK-V50 cells. Photographs are time-lapse images of M-V0 and OSK-V50 cells. The sorted cells were subsequently cultured for five days and then observed every six hours for 42 hr. Time-lapse imaging revealed that there was a higher diversity in both the morphology and mobility in OSK-V50 cells (lower panel) compared to M-V0 cells (upper panel). Original magnification, ×20. Movies of M-V0 and OSK-V50 cells are shown in Video S1 and Video S2 , respectively.

    Journal: PLoS ONE

    Article Title: Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors

    doi: 10.1371/journal.pone.0101735

    Figure Lengend Snippet: Colonic lineage specificity and potency to produce phenotypical diversity in OSK-V50 cells. (A) Immunocytochemistry for CDX2 and CK20 protein. OSK-V50 cells were positive for CDX2 and CK20, as well as M-V0 cells. The cells were counterstained with Hoechst33342. Scale bars: 100 µm. (B) The phenotypical diversity in morphology and mobility of derivatives of OSK-V50 cells. Photographs are time-lapse images of M-V0 and OSK-V50 cells. The sorted cells were subsequently cultured for five days and then observed every six hours for 42 hr. Time-lapse imaging revealed that there was a higher diversity in both the morphology and mobility in OSK-V50 cells (lower panel) compared to M-V0 cells (upper panel). Original magnification, ×20. Movies of M-V0 and OSK-V50 cells are shown in Video S1 and Video S2 , respectively.

    Article Snippet: For immunocytochemistry, the cultured cells, which were fixed with 4% paraformaldehyde, were stained with anti-CK20 antibody (Clone: Ks20.8, dilution 1∶100) or anti-CDX2 antibody (Clone: CDX2-88, prediluted, Abcam), and counterstained with Hoechst33342 (Life Technologies) to identify all nuclei.

    Techniques: Immunocytochemistry, Cell Culture, Imaging

    The histology of the xenografts derived from M-SW480 and OSK-V50 cells. (A) Hematoxylin and eosin staining (HE). The tumors derived from M-SW480 cells predominantly consisted of homogeneous cells. On the other hand, the tumors derived from OSK-V50 cells showed glandular structures. (B) Immunohistochemical analysis. The tumors derived from M-SW480 cells were negative for CK20 and CK7. The tumors derived from OSK-V50 cells were partially positive for CK20 and negative for CK7. Tumors derived from both M-SW480 and OSK-V50 cells were positive for CDX2, but CDX2-negative cells were obviously seen in the tumors of OSK-V50 cells. Scale bars: 50 µm.

    Journal: PLoS ONE

    Article Title: Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors

    doi: 10.1371/journal.pone.0101735

    Figure Lengend Snippet: The histology of the xenografts derived from M-SW480 and OSK-V50 cells. (A) Hematoxylin and eosin staining (HE). The tumors derived from M-SW480 cells predominantly consisted of homogeneous cells. On the other hand, the tumors derived from OSK-V50 cells showed glandular structures. (B) Immunohistochemical analysis. The tumors derived from M-SW480 cells were negative for CK20 and CK7. The tumors derived from OSK-V50 cells were partially positive for CK20 and negative for CK7. Tumors derived from both M-SW480 and OSK-V50 cells were positive for CDX2, but CDX2-negative cells were obviously seen in the tumors of OSK-V50 cells. Scale bars: 50 µm.

    Article Snippet: For immunocytochemistry, the cultured cells, which were fixed with 4% paraformaldehyde, were stained with anti-CK20 antibody (Clone: Ks20.8, dilution 1∶100) or anti-CDX2 antibody (Clone: CDX2-88, prediluted, Abcam), and counterstained with Hoechst33342 (Life Technologies) to identify all nuclei.

    Techniques: Derivative Assay, Staining, Immunohistochemistry

    WNT Establishes Spinal Cord Identity via CDX-Dependent Chromatin Remodeling (A) Proportion of NMP, NMP/SC, or epiblast genomic sites from the SOM ( Figure 2 A) that overlap with accessible regions in vivo ( Neijts et al., 2016 ; this study). 71% of all NMP sites identified in vitro are found in the posterior E7.5 embryo (E7.5-P; Neijts et al., 2016 ). This contrasts with NPs from the spinal cord (E9.5-SC) or anterior nervous system (E9.5-A; this study), which show little overlap with these sites. (B) The average accessibility profile of NMP/SC and NMP-specific sites in wild-type versus T/Bra −/− mutant cells. These sites remain accessible in T/Bra −/− mutant cells at D3 of the spinal cord differentiation. (C) T/Bra −/− mutant cells differentiated to D5 under spinal cord conditions retain accessibility at spinal cord genomic sites. (D) Heatmap showing NMP (top panels) and NMP/SC (bottom panels) site accessibility in D3NMP conditions from WT, T/Bra −/− , and Cdx1/2/4 ( Cdx −/− ) mutant cells. These sites are maintained in the absence of T/Bra but are reduced in the absence of the three Cdx TFs. (E–H) qRT-PCR of Hox genes at D5 indicates AP identity of hindbrain (D5H) and spinal cord (D5SC) cells in wild-type compared with T/Bra −/− and Cdx −/− mutant cells differentiated under spinal cord conditions. Bra −/− mutant cells retain expression of spinal cord Hox genes Hoxb9 (E) and Hoxc6 (F) in contrast to Cdx mutant cells, which express Hoxb4 (G) and Hoxc4 (H). Error bars represent the standard deviation. (I) ChIP-seq enrichment analysis reveals that CDX2 is highly enriched at NMP-specific sites (p values; one-sided Fisher’s exact test; multiple testing corrected using Benjamini-Hochberg procedure). (J) Tn5 insertion frequency, across all SOM regions containing at least one CDX2 motif, at nucleotide resolution in D3NMP cells reveals the presence of a footprint centered on the CDX2 motif. (K) CDX2 (cyan) and Sox2 (red) immunofluorescence at D3. Scale bars represent 20 μm. (L) Histogram of CDX2-positive cells at D3 comparing WT (blue) with Cdx −/− mutant cells (gray), determined by flow cytometry. Numbers indicate percentage of SOX2-positive cells that are CDX2 positive. (M) Removal of the three Cdx transcription factors Cdx1 /2/4 results in the continued induction of OLIG2 (gold) and ectopic production of cranial MN progenitors, marked by PHOX2B (magenta). Scale bars represent 20 μm. (N) The average profile of spinal cord sites (left plot) shows that, relative to D5 spinal cord (D5SC, red), accessibility at these sites is reduced in Cdx −/− mutant cells differentiated under the same conditions (D5SC Cdx −/− , green), to the same extent as D5 hindbrain cells (yellow). Under spinal cord conditions, Cdx mutant cells show increased accessibility at hindbrain sites (right plot).

    Journal: Cell

    Article Title: Nervous System Regionalization Entails Axial Allocation before Neural Differentiation

    doi: 10.1016/j.cell.2018.09.040

    Figure Lengend Snippet: WNT Establishes Spinal Cord Identity via CDX-Dependent Chromatin Remodeling (A) Proportion of NMP, NMP/SC, or epiblast genomic sites from the SOM ( Figure 2 A) that overlap with accessible regions in vivo ( Neijts et al., 2016 ; this study). 71% of all NMP sites identified in vitro are found in the posterior E7.5 embryo (E7.5-P; Neijts et al., 2016 ). This contrasts with NPs from the spinal cord (E9.5-SC) or anterior nervous system (E9.5-A; this study), which show little overlap with these sites. (B) The average accessibility profile of NMP/SC and NMP-specific sites in wild-type versus T/Bra −/− mutant cells. These sites remain accessible in T/Bra −/− mutant cells at D3 of the spinal cord differentiation. (C) T/Bra −/− mutant cells differentiated to D5 under spinal cord conditions retain accessibility at spinal cord genomic sites. (D) Heatmap showing NMP (top panels) and NMP/SC (bottom panels) site accessibility in D3NMP conditions from WT, T/Bra −/− , and Cdx1/2/4 ( Cdx −/− ) mutant cells. These sites are maintained in the absence of T/Bra but are reduced in the absence of the three Cdx TFs. (E–H) qRT-PCR of Hox genes at D5 indicates AP identity of hindbrain (D5H) and spinal cord (D5SC) cells in wild-type compared with T/Bra −/− and Cdx −/− mutant cells differentiated under spinal cord conditions. Bra −/− mutant cells retain expression of spinal cord Hox genes Hoxb9 (E) and Hoxc6 (F) in contrast to Cdx mutant cells, which express Hoxb4 (G) and Hoxc4 (H). Error bars represent the standard deviation. (I) ChIP-seq enrichment analysis reveals that CDX2 is highly enriched at NMP-specific sites (p values; one-sided Fisher’s exact test; multiple testing corrected using Benjamini-Hochberg procedure). (J) Tn5 insertion frequency, across all SOM regions containing at least one CDX2 motif, at nucleotide resolution in D3NMP cells reveals the presence of a footprint centered on the CDX2 motif. (K) CDX2 (cyan) and Sox2 (red) immunofluorescence at D3. Scale bars represent 20 μm. (L) Histogram of CDX2-positive cells at D3 comparing WT (blue) with Cdx −/− mutant cells (gray), determined by flow cytometry. Numbers indicate percentage of SOX2-positive cells that are CDX2 positive. (M) Removal of the three Cdx transcription factors Cdx1 /2/4 results in the continued induction of OLIG2 (gold) and ectopic production of cranial MN progenitors, marked by PHOX2B (magenta). Scale bars represent 20 μm. (N) The average profile of spinal cord sites (left plot) shows that, relative to D5 spinal cord (D5SC, red), accessibility at these sites is reduced in Cdx −/− mutant cells differentiated under the same conditions (D5SC Cdx −/− , green), to the same extent as D5 hindbrain cells (yellow). Under spinal cord conditions, Cdx mutant cells show increased accessibility at hindbrain sites (right plot).

    Article Snippet: Primary antibodies were diluted as follows: mouse anti-CDX2 (1:250, Abcam 157524, RRID: AB_2721036 ), rabbit anti-SOX2 (1:500, Cell Signaling 2748S, RRID: AB_823640 ) and goat anti-SOX1 (1:250, R & D AF3369, RRID: AB_2239879 ); PHOX2B rabbit, kindly provided by Jean-Francois Brunet , RRID: AB_2313690 , 1/1000; OLIG2 rabbit (Millipore, AB9610; RRID: AB_570666 ; 1/1000); Olig2 guinea-pig, kindly provided by Ben Novitch , RRID: AB_2715520 , 1/10000, and SOX2 goat (R & D Systems, AF2018; RRID: AB_355110 ; 1/500).

    Techniques: In Vivo, In Vitro, Mutagenesis, Quantitative RT-PCR, Expressing, Standard Deviation, Chromatin Immunoprecipitation, Immunofluorescence, Flow Cytometry, Cytometry

    CDX2 Can Replace WNT and Prolong Spinal Cord Competency (A) Schematic of the differentiation using iCDX2 ESCs ( Niwa et al., 2005 ) to induce CDX2 between D2 and D3 (SCind). (B) Immunofluorescence of NPs at D5 PHOX2B (cyan) and SOX2 (red). Cranial MNs in hindbrain, but not spinal cord or SCind. (C) qRT-PCR analysis at D5 shows that the induction of CDX2 between D2 and D3 maintains Olig2 expression and upregulates Hoxb9 and Hoxc6 . Error bars represent the standard deviation. (D) Chromatin accessibility, measured by ATAC-seq, at hindbrain (yellow) and spinal cord (red) sites at D5. Spinal cord sites are more open in WT spinal cord cells than WT hindbrain cells (left plot). The induction of CDX2 between D2 and D3 (D5SCind) increases accessibility at spinal cord sites versus D5H WT cells (middle plot) and similar levels of accessibility in hindbrain and spinal cord sites when compared to D5SC WT cells (right plot). (E) Schematic of the differentiation using iCDX2 ESCs to induce CDX2 between D4 and D5 under hindbrain conditions (Hrep) versus hindbrain+ (H+) conditions, in which WNT signaling is activated between D4 and D5. (F and G) qRT-PCR data indicate that, by D5, the induction of CDX2 is sufficient to repress Phox2b but maintain Olig2 (F), in contrast to H + cells, which repress Olig2 expression ( Figure 3 ). The induction of CDX2 between D4 and D5 upregulates posterior Hox genes Hoxb9 , Hoxb8 , and Hoxc6 (G). Error bars represent the standard deviation. (H) Accessibility at hindbrain (yellow) and spinal cord (red) sites reveals that D5Hrep cells display increased accessibility at spinal cord sites and loss of hindbrain sites compared to D5H cells. (I and J) WNT reporter embryos cultured for 14 hr in control versus WNT signaling conditions ( Table S3 ) from E7.0 (I) or E7.5 (J). Images show embryos cultured in media containing bFGF (control) versus FGF and CHIR99021 (FGF/WNT) conditions. Embryos are oriented with anterior at the top of the image. Dashed white lines demarcate the anterior limit. (I) Ventral view of E7.0 cultured embryos. Ectopic induction of WNT activity (n = 10/13) and CDX2 (green, white bracket) is observed in the presence of WNT signaling (n = 10/13), but not control conditions (n = 0/10). SOX2 marks the epiblast (red). Asterisk demarcates the position of the node. Scale bars represent 250 μm. (J) Ectopic induction of WNT signaling in E7.5 cultured embryos (n = 13/16) versus control conditions (n = 0/6). No CDX2 (green) expansion was detected in the anterior neural plate (n = 0/31) marked by SOX1 (red) versus control conditions (n = 0/30). Top panels in (J) show ventral views; bottom panels in (J) show dorsal views. Scale bars represent 250 μm. (K) Chick whole-mount in situ hybridization for Cdx2 following 10-hr ex vivo embryo culture from HH stage 3+ (top panels) and stage 7 (bottom panels). The addition of WNT signals promotes ectopic (white arrows) anterior Cdx2 expression at early stages (15/15 in the FGF/WNT versus n = 0/13 control at stage 3+). By contrast, no expansion is observed in response to WNT signaling in stage 7 embryos that already contain a neural plate (n = 0/9 in FGF/WNT and n = 0/12 in control). White arrowheads mark the anterior limit of Cdx2 expression. Scale bars represent 500 μm. cc, cardiac crescent; mb, midbrain; nt, neural tube; p, posterior.

    Journal: Cell

    Article Title: Nervous System Regionalization Entails Axial Allocation before Neural Differentiation

    doi: 10.1016/j.cell.2018.09.040

    Figure Lengend Snippet: CDX2 Can Replace WNT and Prolong Spinal Cord Competency (A) Schematic of the differentiation using iCDX2 ESCs ( Niwa et al., 2005 ) to induce CDX2 between D2 and D3 (SCind). (B) Immunofluorescence of NPs at D5 PHOX2B (cyan) and SOX2 (red). Cranial MNs in hindbrain, but not spinal cord or SCind. (C) qRT-PCR analysis at D5 shows that the induction of CDX2 between D2 and D3 maintains Olig2 expression and upregulates Hoxb9 and Hoxc6 . Error bars represent the standard deviation. (D) Chromatin accessibility, measured by ATAC-seq, at hindbrain (yellow) and spinal cord (red) sites at D5. Spinal cord sites are more open in WT spinal cord cells than WT hindbrain cells (left plot). The induction of CDX2 between D2 and D3 (D5SCind) increases accessibility at spinal cord sites versus D5H WT cells (middle plot) and similar levels of accessibility in hindbrain and spinal cord sites when compared to D5SC WT cells (right plot). (E) Schematic of the differentiation using iCDX2 ESCs to induce CDX2 between D4 and D5 under hindbrain conditions (Hrep) versus hindbrain+ (H+) conditions, in which WNT signaling is activated between D4 and D5. (F and G) qRT-PCR data indicate that, by D5, the induction of CDX2 is sufficient to repress Phox2b but maintain Olig2 (F), in contrast to H + cells, which repress Olig2 expression ( Figure 3 ). The induction of CDX2 between D4 and D5 upregulates posterior Hox genes Hoxb9 , Hoxb8 , and Hoxc6 (G). Error bars represent the standard deviation. (H) Accessibility at hindbrain (yellow) and spinal cord (red) sites reveals that D5Hrep cells display increased accessibility at spinal cord sites and loss of hindbrain sites compared to D5H cells. (I and J) WNT reporter embryos cultured for 14 hr in control versus WNT signaling conditions ( Table S3 ) from E7.0 (I) or E7.5 (J). Images show embryos cultured in media containing bFGF (control) versus FGF and CHIR99021 (FGF/WNT) conditions. Embryos are oriented with anterior at the top of the image. Dashed white lines demarcate the anterior limit. (I) Ventral view of E7.0 cultured embryos. Ectopic induction of WNT activity (n = 10/13) and CDX2 (green, white bracket) is observed in the presence of WNT signaling (n = 10/13), but not control conditions (n = 0/10). SOX2 marks the epiblast (red). Asterisk demarcates the position of the node. Scale bars represent 250 μm. (J) Ectopic induction of WNT signaling in E7.5 cultured embryos (n = 13/16) versus control conditions (n = 0/6). No CDX2 (green) expansion was detected in the anterior neural plate (n = 0/31) marked by SOX1 (red) versus control conditions (n = 0/30). Top panels in (J) show ventral views; bottom panels in (J) show dorsal views. Scale bars represent 250 μm. (K) Chick whole-mount in situ hybridization for Cdx2 following 10-hr ex vivo embryo culture from HH stage 3+ (top panels) and stage 7 (bottom panels). The addition of WNT signals promotes ectopic (white arrows) anterior Cdx2 expression at early stages (15/15 in the FGF/WNT versus n = 0/13 control at stage 3+). By contrast, no expansion is observed in response to WNT signaling in stage 7 embryos that already contain a neural plate (n = 0/9 in FGF/WNT and n = 0/12 in control). White arrowheads mark the anterior limit of Cdx2 expression. Scale bars represent 500 μm. cc, cardiac crescent; mb, midbrain; nt, neural tube; p, posterior.

    Article Snippet: Primary antibodies were diluted as follows: mouse anti-CDX2 (1:250, Abcam 157524, RRID: AB_2721036 ), rabbit anti-SOX2 (1:500, Cell Signaling 2748S, RRID: AB_823640 ) and goat anti-SOX1 (1:250, R & D AF3369, RRID: AB_2239879 ); PHOX2B rabbit, kindly provided by Jean-Francois Brunet , RRID: AB_2313690 , 1/1000; OLIG2 rabbit (Millipore, AB9610; RRID: AB_570666 ; 1/1000); Olig2 guinea-pig, kindly provided by Ben Novitch , RRID: AB_2715520 , 1/10000, and SOX2 goat (R & D Systems, AF2018; RRID: AB_355110 ; 1/500).

    Techniques: Immunofluorescence, Quantitative RT-PCR, Expressing, Standard Deviation, Cell Culture, Activity Assay, In Situ Hybridization, Ex Vivo, Embryo Culture

    Differential Enhancer Usage Reveals NP Axial Identity (A) Self-organizing map (SOM) of regulatory regions showing differential accessibility relative to D0. Each plot represents the chromatin accessibility Z score for each genomic region in the cluster for each condition (see key in A’). Many sites are common (“neural sites”) to all NPs (black cluster; n = 5,584). These differ from epiblast regulatory regions that are accessible at early stages of the differentiation (Epi; green; n = 1,714). Region-specific sites are identified in anterior (blue; n = 1,863), hindbrain (orange; n = 2,509), and spinal cord (red; n = 1,538) progenitors. A distinct set of regulatory regions identifies D3 NMPs (pink; n = 454 regions). A/H represents shared anterior and hindbrain accessible sites (purple; n = 1,276); H/SC, shared hindbrain and spinal cord sites (lime; n = 1,840); and NMP/SC, shared NMP and spinal cord sites (brown; n = 421). Grey are unclassified sites. (B–D) Examples of ATAC-seq accessible regions that define anterior (B), hindbrain (C), or spinal cord (D) D5 progenitors, identified using the SOM (A). Gene expression from mRNA-seq (error bars = SEM) is shown to the right. Anterior progenitors display region-specific open sites at Shh (B), and hindbrain progenitors demonstrate a Phox2b site (C) and a Hoxc8 site opens in spinal cord (D). (E–G) In vivo ATAC-seq correlates with in vitro . NPs obtained from brain (E; blue shading) and spinal cord (E; red shading) of E9.5 Sox2eGFP embryos. The fold change in accessibility at anterior (blue; n = 1,863) and spinal cord (red; n = 1,538) sites identified in vitro in spinal cord NPs relative to anterior NPs in vivo correlates with AP identity (F). Common neural sites in vitro (black) are similar in both populations in vivo . Anterior sites identified in vitro show preferential accessibility in vivo in anterior NPs (G), and spinal cord in vitro sites show more accessibility in vivo in spinal cord NPs (p values; Wilcoxon rank-sum test). (H–J) ChIP-seq enrichment analysis in anterior (H), hindbrain (I), and spinal cord sites (J). SOX2 ChIP-seq in D5 hindbrain (D5H) and spinal cord (D5SC) cells reveals that the binding site preference is condition specific (I and J). CDX2 ∗ denotes CDX2 ChIP-seq performed in the presence of FGF signaling ( Mazzoni et al., 2013 ). FPM, fragments per million; neural EB, embryoid bodied-derived NPs; NMP, neuromesodermal progenitors; NP, neural progenitors; NT, neural tube; pMN, motor neuron progenitors. See also Figure S2 .

    Journal: Cell

    Article Title: Nervous System Regionalization Entails Axial Allocation before Neural Differentiation

    doi: 10.1016/j.cell.2018.09.040

    Figure Lengend Snippet: Differential Enhancer Usage Reveals NP Axial Identity (A) Self-organizing map (SOM) of regulatory regions showing differential accessibility relative to D0. Each plot represents the chromatin accessibility Z score for each genomic region in the cluster for each condition (see key in A’). Many sites are common (“neural sites”) to all NPs (black cluster; n = 5,584). These differ from epiblast regulatory regions that are accessible at early stages of the differentiation (Epi; green; n = 1,714). Region-specific sites are identified in anterior (blue; n = 1,863), hindbrain (orange; n = 2,509), and spinal cord (red; n = 1,538) progenitors. A distinct set of regulatory regions identifies D3 NMPs (pink; n = 454 regions). A/H represents shared anterior and hindbrain accessible sites (purple; n = 1,276); H/SC, shared hindbrain and spinal cord sites (lime; n = 1,840); and NMP/SC, shared NMP and spinal cord sites (brown; n = 421). Grey are unclassified sites. (B–D) Examples of ATAC-seq accessible regions that define anterior (B), hindbrain (C), or spinal cord (D) D5 progenitors, identified using the SOM (A). Gene expression from mRNA-seq (error bars = SEM) is shown to the right. Anterior progenitors display region-specific open sites at Shh (B), and hindbrain progenitors demonstrate a Phox2b site (C) and a Hoxc8 site opens in spinal cord (D). (E–G) In vivo ATAC-seq correlates with in vitro . NPs obtained from brain (E; blue shading) and spinal cord (E; red shading) of E9.5 Sox2eGFP embryos. The fold change in accessibility at anterior (blue; n = 1,863) and spinal cord (red; n = 1,538) sites identified in vitro in spinal cord NPs relative to anterior NPs in vivo correlates with AP identity (F). Common neural sites in vitro (black) are similar in both populations in vivo . Anterior sites identified in vitro show preferential accessibility in vivo in anterior NPs (G), and spinal cord in vitro sites show more accessibility in vivo in spinal cord NPs (p values; Wilcoxon rank-sum test). (H–J) ChIP-seq enrichment analysis in anterior (H), hindbrain (I), and spinal cord sites (J). SOX2 ChIP-seq in D5 hindbrain (D5H) and spinal cord (D5SC) cells reveals that the binding site preference is condition specific (I and J). CDX2 ∗ denotes CDX2 ChIP-seq performed in the presence of FGF signaling ( Mazzoni et al., 2013 ). FPM, fragments per million; neural EB, embryoid bodied-derived NPs; NMP, neuromesodermal progenitors; NP, neural progenitors; NT, neural tube; pMN, motor neuron progenitors. See also Figure S2 .

    Article Snippet: Primary antibodies were diluted as follows: mouse anti-CDX2 (1:250, Abcam 157524, RRID: AB_2721036 ), rabbit anti-SOX2 (1:500, Cell Signaling 2748S, RRID: AB_823640 ) and goat anti-SOX1 (1:250, R & D AF3369, RRID: AB_2239879 ); PHOX2B rabbit, kindly provided by Jean-Francois Brunet , RRID: AB_2313690 , 1/1000; OLIG2 rabbit (Millipore, AB9610; RRID: AB_570666 ; 1/1000); Olig2 guinea-pig, kindly provided by Ben Novitch , RRID: AB_2715520 , 1/10000, and SOX2 goat (R & D Systems, AF2018; RRID: AB_355110 ; 1/500).

    Techniques: Expressing, In Vivo, In Vitro, Chromatin Immunoprecipitation, Binding Assay, Derivative Assay

    CDX2 Occupancy in Open Chromatin Sites and Associated Gene Ontology Enrichment, Related to Figure 4 (A) The proportion of accessible regions bound by C DX2, as indicated by CDX2 ChIP-seq analysis from neuromesodermal progenitors (NMP, light blue bars, Amin et al., 2016 ) and motor neuron progenitors (pMNs, dark blue bars, Mazzoni et al., 2013 ) derived in vitro , compared with the accessible regions recovered from the self-organizing map (SOM) in this study (refer to Figure 2 A). The overlap demonstrates that CDX2 binds to NMP, NMP/SC (NMP and spinal cord shared) and spinal cord (SC) sites identified by ATAC-seq. Furthermore, in pMN conditions, CDX2 binds accessible regions that are shared between the hindbrain and spinal cord (A’, boxed region outlined in green). CDX2 also targets hindbrain accessible sites (A’’). (A’) The Phox2b genomic region represents a shared hindbrain/spinal cord accessible site that is bound by CDX2 in pMN conditions. (A’’) A hindbrain-accessible site is bound by CDX2 at the Mafb locus in pMN conditions. (B) Region heatmap showing CDX2 binding at open chromatin sites recovered from the SOM (pMN; Mazzoni et al., 2013 ). (C–F) Gene ontology enrichment analysis for CDX2-bound regions shown in (B). In hindbrain accessible regions (D), CDX2 binding is associated with neural genes in contrast to either the NMP and spinal cord (NMP/SC) shared (E) or SC-specific sites (F), which target genes involved in anterior-posterior patterning. (G) Comparison of log2 fold gene expression changes in D5 spinal cord (D5SC) versus D5 hindbrain (D5H), determined by mRNA-seq ( Gouti et al., 2014 ), and wild-type (WT) versus Cdx 2-induced motor neuron progenitors (iCdx2-pMNs) determined by microarray ( Mazzoni et al., 2013 ). CDX2 induction positively correlates with Hoxb9 and other 5′ Hox genes while it negatively correlates with Aldh1a2 in the spinal cord, in agreement with previous studies ( Gouti et al., 2017 ). CDX negatively correlates with hindbrain genes including Phox2b . Color filling indicates –log10(adj. pvalue) from the D5SC versus D5H comparison using DESeq2 ( Love et al., 2014 ). (H) Motif enrichment analysis of CDX2-bound regions shown in (B). Heatmap colors represent the normalized enrichment score computed using iCis Target ( Imrichová et al., 2015 ). CDX2 binds to hindbrain accessible regions that are enriched with SOX factor motifs, in contrast to HOX motifs found in spinal cord sites.

    Journal: Cell

    Article Title: Nervous System Regionalization Entails Axial Allocation before Neural Differentiation

    doi: 10.1016/j.cell.2018.09.040

    Figure Lengend Snippet: CDX2 Occupancy in Open Chromatin Sites and Associated Gene Ontology Enrichment, Related to Figure 4 (A) The proportion of accessible regions bound by C DX2, as indicated by CDX2 ChIP-seq analysis from neuromesodermal progenitors (NMP, light blue bars, Amin et al., 2016 ) and motor neuron progenitors (pMNs, dark blue bars, Mazzoni et al., 2013 ) derived in vitro , compared with the accessible regions recovered from the self-organizing map (SOM) in this study (refer to Figure 2 A). The overlap demonstrates that CDX2 binds to NMP, NMP/SC (NMP and spinal cord shared) and spinal cord (SC) sites identified by ATAC-seq. Furthermore, in pMN conditions, CDX2 binds accessible regions that are shared between the hindbrain and spinal cord (A’, boxed region outlined in green). CDX2 also targets hindbrain accessible sites (A’’). (A’) The Phox2b genomic region represents a shared hindbrain/spinal cord accessible site that is bound by CDX2 in pMN conditions. (A’’) A hindbrain-accessible site is bound by CDX2 at the Mafb locus in pMN conditions. (B) Region heatmap showing CDX2 binding at open chromatin sites recovered from the SOM (pMN; Mazzoni et al., 2013 ). (C–F) Gene ontology enrichment analysis for CDX2-bound regions shown in (B). In hindbrain accessible regions (D), CDX2 binding is associated with neural genes in contrast to either the NMP and spinal cord (NMP/SC) shared (E) or SC-specific sites (F), which target genes involved in anterior-posterior patterning. (G) Comparison of log2 fold gene expression changes in D5 spinal cord (D5SC) versus D5 hindbrain (D5H), determined by mRNA-seq ( Gouti et al., 2014 ), and wild-type (WT) versus Cdx 2-induced motor neuron progenitors (iCdx2-pMNs) determined by microarray ( Mazzoni et al., 2013 ). CDX2 induction positively correlates with Hoxb9 and other 5′ Hox genes while it negatively correlates with Aldh1a2 in the spinal cord, in agreement with previous studies ( Gouti et al., 2017 ). CDX negatively correlates with hindbrain genes including Phox2b . Color filling indicates –log10(adj. pvalue) from the D5SC versus D5H comparison using DESeq2 ( Love et al., 2014 ). (H) Motif enrichment analysis of CDX2-bound regions shown in (B). Heatmap colors represent the normalized enrichment score computed using iCis Target ( Imrichová et al., 2015 ). CDX2 binds to hindbrain accessible regions that are enriched with SOX factor motifs, in contrast to HOX motifs found in spinal cord sites.

    Article Snippet: Primary antibodies were diluted as follows: mouse anti-CDX2 (1:250, Abcam 157524, RRID: AB_2721036 ), rabbit anti-SOX2 (1:500, Cell Signaling 2748S, RRID: AB_823640 ) and goat anti-SOX1 (1:250, R & D AF3369, RRID: AB_2239879 ); PHOX2B rabbit, kindly provided by Jean-Francois Brunet , RRID: AB_2313690 , 1/1000; OLIG2 rabbit (Millipore, AB9610; RRID: AB_570666 ; 1/1000); Olig2 guinea-pig, kindly provided by Ben Novitch , RRID: AB_2715520 , 1/10000, and SOX2 goat (R & D Systems, AF2018; RRID: AB_355110 ; 1/500).

    Techniques: Chromatin Immunoprecipitation, Derivative Assay, In Vitro, Binding Assay, Expressing, Microarray

    Axial Identity Is Established in Cells prior to Neural Identity (A–C) The average accessibility ( Z score) of region-specific sites over time in anterior (labeled “A”), hindbrain (labeled “H”), or spinal cord (labeled “SC”) conditions. AP-specific sites become accessible between D3 and D4. Spinal cord progenitors do not transiently open sites corresponding to anterior (A) or hindbrain (B) identity before opening spinal cord-specific sites (C). (D) Neural sites become accessible in all regions at the same time. Error bars = SD. (E) Schematic of the differentiation (H+ condition). (F–N) qRT-PCR of genes at D3 and D5 following the differentiation of cells to hindbrain (D5H), spinal cord (D5SC), or “hindbrain+” (D5H+) identity. The WNT target Notum (F) is observed following WNT signaling treatment at D3 (D3NMP) and D5 (D5H+). Induction of posterior spinal cord Hox genes Hoxb9 and Hoxc8 is dependent on timing: induction in D3NMP follows D2 to D3 treatment with WNT signals, but not at D5 in D5H+ cells following D4 to D5 treatment with the same signals (G and H). Induction of T/Bra and Cdx2 is dependent on timing, responding to early (D2 to D3), but not late (D4 to D5), treatment with WNT signals (I and J). Late treatment of WNT in the D5H+ condition prevents expression of ventral neural genes Phox2b and Olig2 (K and L, compare D5H to D5H+) while dorsal Pax7 (M) and intermediate Dbx1 (N) neural tube genes are induced. Error bars represent the standard deviation. (O) SOX1 immunofluorescence on D3 versus D4 cells cultured in hindbrain (D3A and D4H) or spinal cord (D3NMP and D4SC) conditions. Scale bars represent 20 μm. (P) Sox1 expression, detected by mRNA-seq ( Gouti et al., 2014 ), at the indicated times and conditions. Error bars = SEM.

    Journal: Cell

    Article Title: Nervous System Regionalization Entails Axial Allocation before Neural Differentiation

    doi: 10.1016/j.cell.2018.09.040

    Figure Lengend Snippet: Axial Identity Is Established in Cells prior to Neural Identity (A–C) The average accessibility ( Z score) of region-specific sites over time in anterior (labeled “A”), hindbrain (labeled “H”), or spinal cord (labeled “SC”) conditions. AP-specific sites become accessible between D3 and D4. Spinal cord progenitors do not transiently open sites corresponding to anterior (A) or hindbrain (B) identity before opening spinal cord-specific sites (C). (D) Neural sites become accessible in all regions at the same time. Error bars = SD. (E) Schematic of the differentiation (H+ condition). (F–N) qRT-PCR of genes at D3 and D5 following the differentiation of cells to hindbrain (D5H), spinal cord (D5SC), or “hindbrain+” (D5H+) identity. The WNT target Notum (F) is observed following WNT signaling treatment at D3 (D3NMP) and D5 (D5H+). Induction of posterior spinal cord Hox genes Hoxb9 and Hoxc8 is dependent on timing: induction in D3NMP follows D2 to D3 treatment with WNT signals, but not at D5 in D5H+ cells following D4 to D5 treatment with the same signals (G and H). Induction of T/Bra and Cdx2 is dependent on timing, responding to early (D2 to D3), but not late (D4 to D5), treatment with WNT signals (I and J). Late treatment of WNT in the D5H+ condition prevents expression of ventral neural genes Phox2b and Olig2 (K and L, compare D5H to D5H+) while dorsal Pax7 (M) and intermediate Dbx1 (N) neural tube genes are induced. Error bars represent the standard deviation. (O) SOX1 immunofluorescence on D3 versus D4 cells cultured in hindbrain (D3A and D4H) or spinal cord (D3NMP and D4SC) conditions. Scale bars represent 20 μm. (P) Sox1 expression, detected by mRNA-seq ( Gouti et al., 2014 ), at the indicated times and conditions. Error bars = SEM.

    Article Snippet: Primary antibodies were diluted as follows: mouse anti-CDX2 (1:250, Abcam 157524, RRID: AB_2721036 ), rabbit anti-SOX2 (1:500, Cell Signaling 2748S, RRID: AB_823640 ) and goat anti-SOX1 (1:250, R & D AF3369, RRID: AB_2239879 ); PHOX2B rabbit, kindly provided by Jean-Francois Brunet , RRID: AB_2313690 , 1/1000; OLIG2 rabbit (Millipore, AB9610; RRID: AB_570666 ; 1/1000); Olig2 guinea-pig, kindly provided by Ben Novitch , RRID: AB_2715520 , 1/10000, and SOX2 goat (R & D Systems, AF2018; RRID: AB_355110 ; 1/500).

    Techniques: Labeling, Quantitative RT-PCR, Expressing, Standard Deviation, Immunofluorescence, Cell Culture

    Elevated OCT3/4 levels and earlier restriction of OCT3/4 to the inner cells in p66Shc knockdown blastocysts. Embryos were categorized according to cell number before analysis: 32–64 cells (scrambled mean ± SD = 51.94 ± 8.33, p66Shc KD mean = 49.60 ± 7.93) or 64–128 cells (scrambled mean = 71.38 ± 5.48, p66Shc KD mean = 74.76 ± 4.46). (A) Representative confocal microscopy images of scrambled and p66Shc knockdown 32–64-cell blastocysts immunostained for CDX2 and OCT3/4. Scale bars are 20 μm. (B) Representative confocal microscopy images of scrambled and p66Shc knockdown 64–128-cell blastocysts. Scale bars are 20 μm. (C) p66Shc knockdown blastocysts have similar CDX2-positive/OCT3/4-positive nuclei compared with scrambled controls in 32–64-cell blastocysts ( n = 19, mean ± SEM, Students' t -test). (D) p66Shc knockdown blastocysts have significantly fewer CDX2-positive/OCT3/4-positive nuclei in 64–128-cell blastocyst ( n = 23, * P

    Journal: Stem Cells and Development

    Article Title: Knockdown of p66Shc Alters Lineage-Associated Transcription Factor Expression in Mouse Blastocysts

    doi: 10.1089/scd.2018.0131

    Figure Lengend Snippet: Elevated OCT3/4 levels and earlier restriction of OCT3/4 to the inner cells in p66Shc knockdown blastocysts. Embryos were categorized according to cell number before analysis: 32–64 cells (scrambled mean ± SD = 51.94 ± 8.33, p66Shc KD mean = 49.60 ± 7.93) or 64–128 cells (scrambled mean = 71.38 ± 5.48, p66Shc KD mean = 74.76 ± 4.46). (A) Representative confocal microscopy images of scrambled and p66Shc knockdown 32–64-cell blastocysts immunostained for CDX2 and OCT3/4. Scale bars are 20 μm. (B) Representative confocal microscopy images of scrambled and p66Shc knockdown 64–128-cell blastocysts. Scale bars are 20 μm. (C) p66Shc knockdown blastocysts have similar CDX2-positive/OCT3/4-positive nuclei compared with scrambled controls in 32–64-cell blastocysts ( n = 19, mean ± SEM, Students' t -test). (D) p66Shc knockdown blastocysts have significantly fewer CDX2-positive/OCT3/4-positive nuclei in 64–128-cell blastocyst ( n = 23, * P

    Article Snippet: Membranes were then incubated in primary antibody diluted in 5% skim milk in TBST overnight at 4°C at the following dilutions: mouse anti-Shc (610879, 1:500; BD Biosciences), rabbit anti-CDX2 (ab76541, 1:500; Abcam), and rabbit anti-EOMES (ab23345, 1:1,000; Abcam).

    Techniques: Confocal Microscopy

    The effect of ZGA on the SCNT embryo quality Schematics of the live‐cell imaging experiments. The embryo imaged from pronuclei until blastocyst stage and transferred to pseudopregnant females. Representative live‐cell images of dynamics MERVL::tdTomato expression during SCNT embryos preimplantation development. The selected images from a series acquired every 15 min. The time after starting observation is shown on the upper right corner of each image. Scale bar, 50 μm. Representative DAPI staining of blastocysts of tdTomato + and tdTomato − SCNT embryos after 115 h of culture in vitro . Scale bar, 50 μm. The tdTomato + and tdTomato − SCNT blastocyst cell numbers were determined by counting the DAPI‐stained cells. Three independent experiment replicates were performed. N, total number of embryos analyzed for each condition. Red bars indicated the mean value. Immunofluorescence images of tdTomato + and tdTomato − blastocysts derived from SCNT. Nanog (ICM) and Cdx2 (TE) were used as lineage markers. Representative images from ≥ 55 embryos analyzed in four independent micromanipulations are shown. Scale bar, 50 μm. Representative image of tdTomato + and tdTomato − SCNT embryos retrieved at E6.5. The tdTomato + SCNT embryo displays normal egg‐cylinder morphology. By contrast, the tdTomato − SCNT embryo shows abnormal morphology. Epi, embryonic epiblast; ExEm, extraembryonic ectoderm; EPC, ectoplacental cone. Scale bar, 50 μm. The bar chart showing the efficiency of attachment to the feeder cells of SCNT blastocysts. The efficiency was calculated based on the total number of blastocysts used for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, SD, n ≥ 3. ** P

    Journal: EMBO Reports

    Article Title: KDM6A and KDM6B play contrasting roles in nuclear transfer embryos revealed by MERVL reporter system

    doi: 10.15252/embr.201846240

    Figure Lengend Snippet: The effect of ZGA on the SCNT embryo quality Schematics of the live‐cell imaging experiments. The embryo imaged from pronuclei until blastocyst stage and transferred to pseudopregnant females. Representative live‐cell images of dynamics MERVL::tdTomato expression during SCNT embryos preimplantation development. The selected images from a series acquired every 15 min. The time after starting observation is shown on the upper right corner of each image. Scale bar, 50 μm. Representative DAPI staining of blastocysts of tdTomato + and tdTomato − SCNT embryos after 115 h of culture in vitro . Scale bar, 50 μm. The tdTomato + and tdTomato − SCNT blastocyst cell numbers were determined by counting the DAPI‐stained cells. Three independent experiment replicates were performed. N, total number of embryos analyzed for each condition. Red bars indicated the mean value. Immunofluorescence images of tdTomato + and tdTomato − blastocysts derived from SCNT. Nanog (ICM) and Cdx2 (TE) were used as lineage markers. Representative images from ≥ 55 embryos analyzed in four independent micromanipulations are shown. Scale bar, 50 μm. Representative image of tdTomato + and tdTomato − SCNT embryos retrieved at E6.5. The tdTomato + SCNT embryo displays normal egg‐cylinder morphology. By contrast, the tdTomato − SCNT embryo shows abnormal morphology. Epi, embryonic epiblast; ExEm, extraembryonic ectoderm; EPC, ectoplacental cone. Scale bar, 50 μm. The bar chart showing the efficiency of attachment to the feeder cells of SCNT blastocysts. The efficiency was calculated based on the total number of blastocysts used for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, SD, n ≥ 3. ** P

    Article Snippet: Embryos were incubated with primary antibodies against H3K27me3 (Millipore, ABE44, USA), H3K27me2 (Abcam, ab24684, USA), H3K4me3 (Abcam, ab213224, USA), H3K9me3 (Abcam, ab176916, USA), HA (Santa Cruz, sc‐7392, USA), MuERVL‐Gag (EpiGentek, A‐2801‐100, USA), Oct4 (Santa Cruz, sc‐8629, USA), Sox2 (Santa Cruz, sc‐17319, USA), Cdx2 (Abcam, ab76541, USA), Nanog (Abcam, ab107156, USA), Ssea1 (Santa Cruz, sc‐21702, USA), E‐cadherin (Abcam, ab40772, USA), Nestin (Santa Cruz, sc‐21247, USA), Brachyury (Santa Cruz, sc‐17745, USA), and Gata4 (Santa Cruz, sc‐1237, USA).

    Techniques: Live Cell Imaging, Expressing, Staining, In Vitro, Immunofluorescence, Derivative Assay

    Relative expression of SLC6A1 and CDX2 mRNA and protein. (A) SLC6A1 and CDX2 mRNA was detected in A2780, OVCAR3 and SK-OV-3 ovarian carcinoma cells. SK-OV-3 cells were used for further experiments. Silencing CDX2 downregulated the expression of SLC6A1 (B) protein and (C) mRNA. (D) Silencing SLC6A1 downregulated the expression of SLC6A1 mRNA. Inhibiting miR133a reversed the effect. Results are mean ± standard error of the mean. *P

    Journal: Oncology Letters

    Article Title: SLC6A1-miR133a-CDX2 loop regulates SK-OV-3 ovarian cancer cell proliferation, migration and invasion

    doi: 10.3892/ol.2018.9273

    Figure Lengend Snippet: Relative expression of SLC6A1 and CDX2 mRNA and protein. (A) SLC6A1 and CDX2 mRNA was detected in A2780, OVCAR3 and SK-OV-3 ovarian carcinoma cells. SK-OV-3 cells were used for further experiments. Silencing CDX2 downregulated the expression of SLC6A1 (B) protein and (C) mRNA. (D) Silencing SLC6A1 downregulated the expression of SLC6A1 mRNA. Inhibiting miR133a reversed the effect. Results are mean ± standard error of the mean. *P

    Article Snippet: The primary antibodies used included monoclonal rabbit anti-SLC6A1 (dilution, 1:5,000; DCABH-6396; Creative-Diagnostics, Shirley, USA), monoclonal rabbit anti-CDX2 (dilution, 1:5,000; ab76541, Abcam) and polyclonal rabbit anti-β-actin (dilution, 1:5,000; ab8226, Abcam).

    Techniques: Expressing

    Association between SLC6A1 and CDX2 in SK-OV-3 cells. (A) miR-133a mimics inhibited the mRNA expression of SLC6A1 and CDX2. (B) miR-133a inhibitors upregulated the mRNA expression of SLC6A1 and CDX2. miR-133a mimics reduced the relative luciferase activity of the (C) SLC6A1 and (D) CDX2 3′UTRs compared with mutant 3′UTRs. (E) Silencing CDX2 or SLC6A1 with specific siRNAs upregulated miR-133a expression. Results are mean ± standard error of the mean. *P

    Journal: Oncology Letters

    Article Title: SLC6A1-miR133a-CDX2 loop regulates SK-OV-3 ovarian cancer cell proliferation, migration and invasion

    doi: 10.3892/ol.2018.9273

    Figure Lengend Snippet: Association between SLC6A1 and CDX2 in SK-OV-3 cells. (A) miR-133a mimics inhibited the mRNA expression of SLC6A1 and CDX2. (B) miR-133a inhibitors upregulated the mRNA expression of SLC6A1 and CDX2. miR-133a mimics reduced the relative luciferase activity of the (C) SLC6A1 and (D) CDX2 3′UTRs compared with mutant 3′UTRs. (E) Silencing CDX2 or SLC6A1 with specific siRNAs upregulated miR-133a expression. Results are mean ± standard error of the mean. *P

    Article Snippet: The primary antibodies used included monoclonal rabbit anti-SLC6A1 (dilution, 1:5,000; DCABH-6396; Creative-Diagnostics, Shirley, USA), monoclonal rabbit anti-CDX2 (dilution, 1:5,000; ab76541, Abcam) and polyclonal rabbit anti-β-actin (dilution, 1:5,000; ab8226, Abcam).

    Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis

    Confluence-dependent upregulation of transcription factors in HCT-116 and HCT-15 cells . Culture conditions were the same as for Figure 1. Protein expression was analyzed by Western blotting as described in Materials and Methods. Data shown are representative of three independent experiments. All transcription factors exhibited altered protein expression levels in a confluence-dependent manner except for HNF-1α, which was unchanged. * N/D , Cdx2 was not detected in HCT-116.

    Journal: BMC Cancer

    Article Title: Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines

    doi: 10.1186/1471-2407-11-51

    Figure Lengend Snippet: Confluence-dependent upregulation of transcription factors in HCT-116 and HCT-15 cells . Culture conditions were the same as for Figure 1. Protein expression was analyzed by Western blotting as described in Materials and Methods. Data shown are representative of three independent experiments. All transcription factors exhibited altered protein expression levels in a confluence-dependent manner except for HNF-1α, which was unchanged. * N/D , Cdx2 was not detected in HCT-116.

    Article Snippet: After blocking with 5% nonfat milk in 0.1% Tween 20-TBS for 1 hour at room temperature, membranes were incubated with the appropriate primary antibodies in 1% blocking buffer for 2 hours at room temperature or overnight at 4°C [anti-CD26 (MI1004, Calbiochem, San Diego, CA or AF1180, R & D Systems, Minneapolis, MN), anti-Cdx2 (MU392A-UC, BioGenex, San Ramon, CA), anti-α-tubulin (T9026, Sigma, Saint Louis, MO), anti-c-Myc (9E10), anti-USF-1 (C-20), anti-HNF-1α (C-19) (sc-40, sc-229, and sc-6547, respectively, Santa Cruz, Santa Cruz, CA)], anti-HIF-1α (610958, BD Transduction Laboratories, San Jose, CA)], followed by incubation with the appropriate HRP-conjugated secondary antibodies (Pierce) for 1 hour at room temperature.

    Techniques: Expressing, Western Blot

    Effect of Cdx2 siRNA on confluence-dependent CD26 expression in HCT-15 cells . The effect of Cdx2 siRNA on the confluence-dependent CD26 protein expression in HCT-15 was evaluated following transient transfection (when cells were at 90% confluence) of control siRNA ( left ) or Cdx2 siRNA ( right ) as described in Materials and Methods. CD26 and Cdx2 protein expression were analyzed by Western blotting and quantified by densitometric analysis. Data shown are representative of three independent experiments.

    Journal: BMC Cancer

    Article Title: Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines

    doi: 10.1186/1471-2407-11-51

    Figure Lengend Snippet: Effect of Cdx2 siRNA on confluence-dependent CD26 expression in HCT-15 cells . The effect of Cdx2 siRNA on the confluence-dependent CD26 protein expression in HCT-15 was evaluated following transient transfection (when cells were at 90% confluence) of control siRNA ( left ) or Cdx2 siRNA ( right ) as described in Materials and Methods. CD26 and Cdx2 protein expression were analyzed by Western blotting and quantified by densitometric analysis. Data shown are representative of three independent experiments.

    Article Snippet: After blocking with 5% nonfat milk in 0.1% Tween 20-TBS for 1 hour at room temperature, membranes were incubated with the appropriate primary antibodies in 1% blocking buffer for 2 hours at room temperature or overnight at 4°C [anti-CD26 (MI1004, Calbiochem, San Diego, CA or AF1180, R & D Systems, Minneapolis, MN), anti-Cdx2 (MU392A-UC, BioGenex, San Ramon, CA), anti-α-tubulin (T9026, Sigma, Saint Louis, MO), anti-c-Myc (9E10), anti-USF-1 (C-20), anti-HNF-1α (C-19) (sc-40, sc-229, and sc-6547, respectively, Santa Cruz, Santa Cruz, CA)], anti-HIF-1α (610958, BD Transduction Laboratories, San Jose, CA)], followed by incubation with the appropriate HRP-conjugated secondary antibodies (Pierce) for 1 hour at room temperature.

    Techniques: Expressing, Transfection, Western Blot

    Distinct Roles of Fgfr1 and Fgfr2 in Primitive Endoderm Development in 129 (A, B) and B6 (C, D) Genetic Backgrounds ( A ) Localization of Nanog (green), Gata4 (red) and Cdx2 (cyan) in wild-type and Fgfr1 −/− ; Fgfr2 −/− ; Zp3 +/Cre embryos on 129 genetic background. Note that a single copy of either Fgfr1 (R1 +/− ; R2 −/− ) or Fgfr2 (R1 −/− ; R2 +/− ) rescues primitive endoderm development. Embryos on the 129 genetic background contained a copy of Zp3 Cre allele ( A , B ). ( B ) Quantitative analysis of number of Nanog + (Epi) and Gata4 + (PrE) cells in Fgfr1 and Fgfr2 compound mutants. Note complete lack of Gata4 + cells in all double-null mutant embryos. A single allele of Fgfr1 was more efficient in rescuing primitive endoderm formation compared to a single allele of Fgfr2 . ( C ) Similar to embryos on the 129 genetic background, all Fgfr1 −/− ; Fgfr2 −/− embryos on the B6 background lack primitive endoderm indicated by loss of Gata4 + cells. Note that a single copy of either receptor rescues primitive endoderm formation. ( D ) On the B6 genetic background, Fgfr1 still plays a primary role in primitive endoderm development, while Fgfr2 contributes more significantly than in the 129 background. Data are represented as mean ± SEM. ( E ) Individual roles of Fgfr1 and Fgfr2 in primitive endoderm development in embryos on 129 and B6 genetic backgrounds. Total number of the embryos and present of embryos without PrE (Gata4 − ).

    Journal: Developmental cell

    Article Title: Distinct Requirements for Fgfr1 and Fgfr2 in Primitive Endoderm Development and Exit from Pluripotency

    doi: 10.1016/j.devcel.2017.05.004

    Figure Lengend Snippet: Distinct Roles of Fgfr1 and Fgfr2 in Primitive Endoderm Development in 129 (A, B) and B6 (C, D) Genetic Backgrounds ( A ) Localization of Nanog (green), Gata4 (red) and Cdx2 (cyan) in wild-type and Fgfr1 −/− ; Fgfr2 −/− ; Zp3 +/Cre embryos on 129 genetic background. Note that a single copy of either Fgfr1 (R1 +/− ; R2 −/− ) or Fgfr2 (R1 −/− ; R2 +/− ) rescues primitive endoderm development. Embryos on the 129 genetic background contained a copy of Zp3 Cre allele ( A , B ). ( B ) Quantitative analysis of number of Nanog + (Epi) and Gata4 + (PrE) cells in Fgfr1 and Fgfr2 compound mutants. Note complete lack of Gata4 + cells in all double-null mutant embryos. A single allele of Fgfr1 was more efficient in rescuing primitive endoderm formation compared to a single allele of Fgfr2 . ( C ) Similar to embryos on the 129 genetic background, all Fgfr1 −/− ; Fgfr2 −/− embryos on the B6 background lack primitive endoderm indicated by loss of Gata4 + cells. Note that a single copy of either receptor rescues primitive endoderm formation. ( D ) On the B6 genetic background, Fgfr1 still plays a primary role in primitive endoderm development, while Fgfr2 contributes more significantly than in the 129 background. Data are represented as mean ± SEM. ( E ) Individual roles of Fgfr1 and Fgfr2 in primitive endoderm development in embryos on 129 and B6 genetic backgrounds. Total number of the embryos and present of embryos without PrE (Gata4 − ).

    Article Snippet: Primary antibodies used were: Nanog (RCAB0002P-F, Cosmo Bio Co., Ltd.), Cdx2 (MU392A-UC, BioGenex), Gata6 (AF1700, R & D Systems), Gata4 (sc-1237, Santa Cruz), Sox17 (AF1924, R & D Systems).

    Techniques: Mutagenesis

    Exogenous Fgf4 Fails to Rescue PrE Formation in Fgfr1 −/− ; Fgfr2 −/− Embryos on 129 and B6 Genetic Backgrounds ( A ) Time-line of the experiments. Embryos were dissected at E2.5 and were cultured in DMEM in the presence of Fgf4 (1000 ng/ml) and heparin (1 μg/ml) for 72h. ( B ) Localizations of Nanog (green), Gata4 (red) and Cdx2 (cyan) positive cells in Fgfr1 +/+ ; Fgfr2 +/+ , Fgfr1 −/− ; Fgfr2 −/− , Fgfr1 +/− ; Fgfr2 −/− and Fgfr1 −/− ; Fgfr2 +/− blastocysts are shown. Note that embryos on the 129 genetic background contained a copy of Zp3 Cre allele. ( C, D ) Quantification of number of Nanog + (Epi) and Gata4 + (PrE) cells in Fgfr1 and Fgfr2 compound mutants in embryos on 129 ( C ) and B6 ( D ) genetic backgrounds. Note stimulation with Fgf4 converted all ICM cells of Fgfr1 +/+ ; Fgfr2 +/+ blastocysts to Gata4 + PrE cells, while exogenous Fgf4 had no effect on the fate of the ICM cells in Fgfr1 −/− ; Fgfr2 −/− blastocysts. Fgf4 stimulation was considerably more potent in embryos with a single wild-type allele of Fgfr1 than Fgfr2. Embryos on the 129 genetic background contained a copy of Zp3 Cre allele. (E) Effect of exogenous Fgf4 (1000 ng/ml) on primitive endoderm development in Fgfr1 and Fgfr2 compound mutants. Total number of embryos and percent of embryos without epiblast (Nanog − ) are shown. ( F ) Effect of exogenous Fgf4 on primitive endoderm development in Fgfr1 +/+ (wild type), Fgfr1 +/ − and Fgfr1 −/− embryos. Percentage of embryos without Epi cells and number of embryos (in parenthesis) are shown. Note that these embryos are wild-type for Fgfr2 . (G) Exogenous Fgf4 does not convert Epi cells into PrE cells in mouse embryos deficient for both copies of Fgfr1 . Percent of embryos without Epi cells is shown. ( H, I) Number of Epi ( H ) and PrE ( I ) cells in wild-type , Fgfr1 +/− ; Fgfr2 +/+ and Fgfr1 −/− ; Fgfr2 +/+ untreated embryos and embryos treated with Fgf4 (500 – 4000 ng/ml). Note Fgf4 at 500 and 1000 ng/ml increased the number of PrE cells in Fgfr1 −/− ; Fgfr2 +/+ embryos ( I ) while the number of Epi cells was not decreased ( H ). Fgf4 at 2000 and 4000 ng/ml decreased the number of PrE cells in treated embryos. Data are represented as mean ± SEM.

    Journal: Developmental cell

    Article Title: Distinct Requirements for Fgfr1 and Fgfr2 in Primitive Endoderm Development and Exit from Pluripotency

    doi: 10.1016/j.devcel.2017.05.004

    Figure Lengend Snippet: Exogenous Fgf4 Fails to Rescue PrE Formation in Fgfr1 −/− ; Fgfr2 −/− Embryos on 129 and B6 Genetic Backgrounds ( A ) Time-line of the experiments. Embryos were dissected at E2.5 and were cultured in DMEM in the presence of Fgf4 (1000 ng/ml) and heparin (1 μg/ml) for 72h. ( B ) Localizations of Nanog (green), Gata4 (red) and Cdx2 (cyan) positive cells in Fgfr1 +/+ ; Fgfr2 +/+ , Fgfr1 −/− ; Fgfr2 −/− , Fgfr1 +/− ; Fgfr2 −/− and Fgfr1 −/− ; Fgfr2 +/− blastocysts are shown. Note that embryos on the 129 genetic background contained a copy of Zp3 Cre allele. ( C, D ) Quantification of number of Nanog + (Epi) and Gata4 + (PrE) cells in Fgfr1 and Fgfr2 compound mutants in embryos on 129 ( C ) and B6 ( D ) genetic backgrounds. Note stimulation with Fgf4 converted all ICM cells of Fgfr1 +/+ ; Fgfr2 +/+ blastocysts to Gata4 + PrE cells, while exogenous Fgf4 had no effect on the fate of the ICM cells in Fgfr1 −/− ; Fgfr2 −/− blastocysts. Fgf4 stimulation was considerably more potent in embryos with a single wild-type allele of Fgfr1 than Fgfr2. Embryos on the 129 genetic background contained a copy of Zp3 Cre allele. (E) Effect of exogenous Fgf4 (1000 ng/ml) on primitive endoderm development in Fgfr1 and Fgfr2 compound mutants. Total number of embryos and percent of embryos without epiblast (Nanog − ) are shown. ( F ) Effect of exogenous Fgf4 on primitive endoderm development in Fgfr1 +/+ (wild type), Fgfr1 +/ − and Fgfr1 −/− embryos. Percentage of embryos without Epi cells and number of embryos (in parenthesis) are shown. Note that these embryos are wild-type for Fgfr2 . (G) Exogenous Fgf4 does not convert Epi cells into PrE cells in mouse embryos deficient for both copies of Fgfr1 . Percent of embryos without Epi cells is shown. ( H, I) Number of Epi ( H ) and PrE ( I ) cells in wild-type , Fgfr1 +/− ; Fgfr2 +/+ and Fgfr1 −/− ; Fgfr2 +/+ untreated embryos and embryos treated with Fgf4 (500 – 4000 ng/ml). Note Fgf4 at 500 and 1000 ng/ml increased the number of PrE cells in Fgfr1 −/− ; Fgfr2 +/+ embryos ( I ) while the number of Epi cells was not decreased ( H ). Fgf4 at 2000 and 4000 ng/ml decreased the number of PrE cells in treated embryos. Data are represented as mean ± SEM.

    Article Snippet: Primary antibodies used were: Nanog (RCAB0002P-F, Cosmo Bio Co., Ltd.), Cdx2 (MU392A-UC, BioGenex), Gata6 (AF1700, R & D Systems), Gata4 (sc-1237, Santa Cruz), Sox17 (AF1924, R & D Systems).

    Techniques: Cell Culture

    Fgfr1-Cerulean and Fgfr2-mCherry Expression in Preimplantation Development ( A ) Schematic representation of Fgfr1-Cerulean and Fgfr2-mCherry reporter alleles. ( B ) Fgfr2-mCherry (red) is detected by direct fluorescence (df) in primitive endoderm (arrowhead) and trophectoderm (arrow) at E3.5. Note absence of Fgfr2-mCherry in epiblast (asterisk). ( C ) Fgfr1-Cerulean (blue) is detected by direct fluorescence (df) in all cell lineages at E3.5. Expression of Nanog (green, epiblast), Gata4 (red, primitive endoderm) and Cdx2 (white, trophectoderm) are shown; arrowhead, PrE. (D) Fgfr2-mCherry expression is detected in all cells of 8-cell embryos at E2.5 by labeling with antibodies (ab) to mCherry (green) and by direct fluorescence (df; red). Note that Cdx2 (cyan) is expressed in all cells at this stage. ( E ) At E3.25 Fgfr2-mCherry is strongly expressed in Cdx2 (cyan) + TE cells while no Fgfr2-mCherry expression is detected in ICM cells by direct fluorescence. Note weak Fgfr2-mCherry labeling in ICM detected with antibodies. ( F ) At E3.25, at the onset of blastocoel development (asterisk), strong Fgfr2-mCherry was detected in Cdx2 + TE cells, while weak homogeneous Fgfr2-mCherry staining was detected in all ICM cells by antibody labeling. Note that no direct Fgfr2-mCherry fluorescence was detected in the ICM cells at this stage. ( G ) At E3.5 strong Fgfr2-mCherry expression was detected in subpopulation of ICM, possibly destined to become PrE cells (arrow). Note that weak direct Fgfr2-mCherry fluorescence is detected in developing PrE cells at this stage (arrow). ( H, I ) Fgfr2-mCherry expression strongly correlates with the Gata6 expression both in ICM and TE cells. ( H ) E3.5 blastocysts were stained with antibodies to mCherry (ab, green) and Gata6 (white); red shows direct mCherry fluorescence (df). DAPI (blue) was used to label nuclei. Individual channel images are shown. ( I ). Note that ICM cells (top panel) are split into two populations: cells that are either negative or have weak Gata6/Fgfr2-mCherry expression (marked with yellow circle), and cells that express high levels of Gata6 and Fgfr2-mCherry . Note that TE cells have considerably higher Fgfr2-mCherry and Gata6 expression when compared to ICM cells, which is reflected by different X-axis scaling. ).

    Journal: Developmental cell

    Article Title: Distinct Requirements for Fgfr1 and Fgfr2 in Primitive Endoderm Development and Exit from Pluripotency

    doi: 10.1016/j.devcel.2017.05.004

    Figure Lengend Snippet: Fgfr1-Cerulean and Fgfr2-mCherry Expression in Preimplantation Development ( A ) Schematic representation of Fgfr1-Cerulean and Fgfr2-mCherry reporter alleles. ( B ) Fgfr2-mCherry (red) is detected by direct fluorescence (df) in primitive endoderm (arrowhead) and trophectoderm (arrow) at E3.5. Note absence of Fgfr2-mCherry in epiblast (asterisk). ( C ) Fgfr1-Cerulean (blue) is detected by direct fluorescence (df) in all cell lineages at E3.5. Expression of Nanog (green, epiblast), Gata4 (red, primitive endoderm) and Cdx2 (white, trophectoderm) are shown; arrowhead, PrE. (D) Fgfr2-mCherry expression is detected in all cells of 8-cell embryos at E2.5 by labeling with antibodies (ab) to mCherry (green) and by direct fluorescence (df; red). Note that Cdx2 (cyan) is expressed in all cells at this stage. ( E ) At E3.25 Fgfr2-mCherry is strongly expressed in Cdx2 (cyan) + TE cells while no Fgfr2-mCherry expression is detected in ICM cells by direct fluorescence. Note weak Fgfr2-mCherry labeling in ICM detected with antibodies. ( F ) At E3.25, at the onset of blastocoel development (asterisk), strong Fgfr2-mCherry was detected in Cdx2 + TE cells, while weak homogeneous Fgfr2-mCherry staining was detected in all ICM cells by antibody labeling. Note that no direct Fgfr2-mCherry fluorescence was detected in the ICM cells at this stage. ( G ) At E3.5 strong Fgfr2-mCherry expression was detected in subpopulation of ICM, possibly destined to become PrE cells (arrow). Note that weak direct Fgfr2-mCherry fluorescence is detected in developing PrE cells at this stage (arrow). ( H, I ) Fgfr2-mCherry expression strongly correlates with the Gata6 expression both in ICM and TE cells. ( H ) E3.5 blastocysts were stained with antibodies to mCherry (ab, green) and Gata6 (white); red shows direct mCherry fluorescence (df). DAPI (blue) was used to label nuclei. Individual channel images are shown. ( I ). Note that ICM cells (top panel) are split into two populations: cells that are either negative or have weak Gata6/Fgfr2-mCherry expression (marked with yellow circle), and cells that express high levels of Gata6 and Fgfr2-mCherry . Note that TE cells have considerably higher Fgfr2-mCherry and Gata6 expression when compared to ICM cells, which is reflected by different X-axis scaling. ).

    Article Snippet: Primary antibodies used were: Nanog (RCAB0002P-F, Cosmo Bio Co., Ltd.), Cdx2 (MU392A-UC, BioGenex), Gata6 (AF1700, R & D Systems), Gata4 (sc-1237, Santa Cruz), Sox17 (AF1924, R & D Systems).

    Techniques: Expressing, Fluorescence, Labeling, Staining, Antibody Labeling

    Model for dynamic CDX2 function during intestinal differentiation In proliferating cells, CDX2 binds with relative selectivity, frequently accompanied by GATA6, at H3K4Me2 regions with selectively open chromatin at loci active in progenitor cells. Genes associated with epithelial maturity are not expressed in these cells and their enhancers have a relatively closed structure with less H3K4Me2. As cells differentiate, CDX2 relocates from progenitor-specific target genes to those associated with maturity, now accompanied by HNF4A; the appearance of an active H3K4Me2 pattern at many loci depends on CDX2.

    Journal: Developmental cell

    Article Title: Differentiation-specific histone modifications reveal dynamic chromatin interactions and alternative partners for the intestinal transcription factor CDX2

    doi: 10.1016/j.devcel.2010.10.006

    Figure Lengend Snippet: Model for dynamic CDX2 function during intestinal differentiation In proliferating cells, CDX2 binds with relative selectivity, frequently accompanied by GATA6, at H3K4Me2 regions with selectively open chromatin at loci active in progenitor cells. Genes associated with epithelial maturity are not expressed in these cells and their enhancers have a relatively closed structure with less H3K4Me2. As cells differentiate, CDX2 relocates from progenitor-specific target genes to those associated with maturity, now accompanied by HNF4A; the appearance of an active H3K4Me2 pattern at many loci depends on CDX2.

    Article Snippet: Co-immunoprecipitation was done using standard techniques and anti-CDX2 (BD Pharmingen 560171; Extended Experimental Procedures).

    Techniques:

    CDX2 interacts dynamically with the genome during intestinal cell differentiation A, Venn diagram representation of binding sites identified by CDX2 ChIP-seq in proliferating and mature Caco-2 cells. B, . C, Condition-specific CDX2 binding corresponds to condition-specific active chromatin. H3K4Me2-marked putative cis -elements were ranked from most differentially active chromatin signatures in proliferating (left) to differentiated cells (right) and plotted along the x-axis in bins of 750. CDX2 occupancy was then plotted relative to occupancy in areas where active chromatin did not vary (center). CDX2 binding sites specific to proliferating (blue triangles) or differentiated cells (red squares) correlated with proliferating and differentiated cell-specific active chromatin, respectively. D, Heat maps demonstrating correlation of stage-specific CDX2 occupancy with stage-specific gene expression. Top: Early (left, blue) versus late (right, red) genes were ranked and binned in groups of 100; genes with unaltered expression are represented in the center. The color map (blue - red) presents the scale of log 2 fold change (−1 to +1) in gene expression levels in the two stages. Bottom: The frequency of condition-specific CDX2 binding within 100 kb of each gene expression bin is indicated by the intensity of yellow shading. Numbers below the color map correspond to the minimum, mean, and maximum ratios (0.5, 1, and 2.4, respectively) between the average number of CDX2 binding sites within 100kb from the genes in the same bin over the average number of binding sites near all genes. E, .

    Journal: Developmental cell

    Article Title: Differentiation-specific histone modifications reveal dynamic chromatin interactions and alternative partners for the intestinal transcription factor CDX2

    doi: 10.1016/j.devcel.2010.10.006

    Figure Lengend Snippet: CDX2 interacts dynamically with the genome during intestinal cell differentiation A, Venn diagram representation of binding sites identified by CDX2 ChIP-seq in proliferating and mature Caco-2 cells. B, . C, Condition-specific CDX2 binding corresponds to condition-specific active chromatin. H3K4Me2-marked putative cis -elements were ranked from most differentially active chromatin signatures in proliferating (left) to differentiated cells (right) and plotted along the x-axis in bins of 750. CDX2 occupancy was then plotted relative to occupancy in areas where active chromatin did not vary (center). CDX2 binding sites specific to proliferating (blue triangles) or differentiated cells (red squares) correlated with proliferating and differentiated cell-specific active chromatin, respectively. D, Heat maps demonstrating correlation of stage-specific CDX2 occupancy with stage-specific gene expression. Top: Early (left, blue) versus late (right, red) genes were ranked and binned in groups of 100; genes with unaltered expression are represented in the center. The color map (blue - red) presents the scale of log 2 fold change (−1 to +1) in gene expression levels in the two stages. Bottom: The frequency of condition-specific CDX2 binding within 100 kb of each gene expression bin is indicated by the intensity of yellow shading. Numbers below the color map correspond to the minimum, mean, and maximum ratios (0.5, 1, and 2.4, respectively) between the average number of CDX2 binding sites within 100kb from the genes in the same bin over the average number of binding sites near all genes. E, .

    Article Snippet: Co-immunoprecipitation was done using standard techniques and anti-CDX2 (BD Pharmingen 560171; Extended Experimental Procedures).

    Techniques: Cell Differentiation, Binding Assay, Chromatin Immunoprecipitation, Expressing

    Cdx2 is required for proliferation and differentiation of the adult mouse intestinal epithelium A, Immunostaining verifies absence of intestinal Cdx2 expression in tamoxifen-treated Cdx2 fV/fV ;Villin-Cre ER(T2) ). B, Reduced mRNA levels of Cdx2 and candidate target genes Sis and Isx in tamoxifen-treated Cdx2 fV/fV ;Villin-Cre ER(T2) intestines; Cdx1 transcript levels are unaffected. C, Cdx2 fV/fV ;Cre ER(T2) mice rapidly lose weight after tamoxifen treatment. D, Intestines of Cdx2 fV/fV ;Villin-Cre ER(T2) mice are dilated and the cecum is particularly engorged with fluid. E, H E staining reveals intact tissue architecture in the Cdx2 mutant ileum. F, A profound deficiency is evident in Alkaline Phosphatase activity, a marker of enterocytes, especially in the distal small intestine. G–I, Periodic acid-Schiff (G), Lysozyme (H) and ChromograninA (I) were used as markers of goblet, Paneth, and enteroendocrine cells, respectively, in the ileum. J, In contrast to the respective single gene mutants, Cdx1 −/− ;Cdx2 fV/fV ;Villin-Cre ER(T2) intestinal crypts show markedly reduced expression of the proliferative markers Ki67 and Pcna. Quantitation of the Ki67 + fraction of crypt cells showed significant reduction in the double mutant (P

    Journal: Developmental cell

    Article Title: Differentiation-specific histone modifications reveal dynamic chromatin interactions and alternative partners for the intestinal transcription factor CDX2

    doi: 10.1016/j.devcel.2010.10.006

    Figure Lengend Snippet: Cdx2 is required for proliferation and differentiation of the adult mouse intestinal epithelium A, Immunostaining verifies absence of intestinal Cdx2 expression in tamoxifen-treated Cdx2 fV/fV ;Villin-Cre ER(T2) ). B, Reduced mRNA levels of Cdx2 and candidate target genes Sis and Isx in tamoxifen-treated Cdx2 fV/fV ;Villin-Cre ER(T2) intestines; Cdx1 transcript levels are unaffected. C, Cdx2 fV/fV ;Cre ER(T2) mice rapidly lose weight after tamoxifen treatment. D, Intestines of Cdx2 fV/fV ;Villin-Cre ER(T2) mice are dilated and the cecum is particularly engorged with fluid. E, H E staining reveals intact tissue architecture in the Cdx2 mutant ileum. F, A profound deficiency is evident in Alkaline Phosphatase activity, a marker of enterocytes, especially in the distal small intestine. G–I, Periodic acid-Schiff (G), Lysozyme (H) and ChromograninA (I) were used as markers of goblet, Paneth, and enteroendocrine cells, respectively, in the ileum. J, In contrast to the respective single gene mutants, Cdx1 −/− ;Cdx2 fV/fV ;Villin-Cre ER(T2) intestinal crypts show markedly reduced expression of the proliferative markers Ki67 and Pcna. Quantitation of the Ki67 + fraction of crypt cells showed significant reduction in the double mutant (P

    Article Snippet: Co-immunoprecipitation was done using standard techniques and anti-CDX2 (BD Pharmingen 560171; Extended Experimental Procedures).

    Techniques: Immunostaining, Expressing, Mouse Assay, Staining, Mutagenesis, Activity Assay, Marker, Quantitation Assay

    Condition-specific CDX2 binding is accompanied by GATA6 in proliferating cells and HNF4A in differentiated cells A . B , Frequency of co-occupancy among TFs in proliferating and differentiated cells as a function of the distance between peak factor-binding sites. Condition-specific CDX2 binding sites more frequently lie near GATA6-bound regions in proliferating cells and near HNF4A-occupied regions in mature cells. The frequency of overlap in binding peaks within a 300bp distance (dotted line) is indicated in pie charts (right). C, Heat maps of all condition-specific CDX2-bound sites, showing the extent of GATA6 and HNF4A co-occupancy at these regions and confirming the relationship between these factors in binding across the genome. Sites are ordered by robustness of CDX2 occupancy in each state of differentiation. The measure of each TF’s occupancy was defined as the relative ChIP signal in relation to the input signal at binding sites (see Experimental Procedures for mathematical details) and distributed between −4 and 12 (color map scale).

    Journal: Developmental cell

    Article Title: Differentiation-specific histone modifications reveal dynamic chromatin interactions and alternative partners for the intestinal transcription factor CDX2

    doi: 10.1016/j.devcel.2010.10.006

    Figure Lengend Snippet: Condition-specific CDX2 binding is accompanied by GATA6 in proliferating cells and HNF4A in differentiated cells A . B , Frequency of co-occupancy among TFs in proliferating and differentiated cells as a function of the distance between peak factor-binding sites. Condition-specific CDX2 binding sites more frequently lie near GATA6-bound regions in proliferating cells and near HNF4A-occupied regions in mature cells. The frequency of overlap in binding peaks within a 300bp distance (dotted line) is indicated in pie charts (right). C, Heat maps of all condition-specific CDX2-bound sites, showing the extent of GATA6 and HNF4A co-occupancy at these regions and confirming the relationship between these factors in binding across the genome. Sites are ordered by robustness of CDX2 occupancy in each state of differentiation. The measure of each TF’s occupancy was defined as the relative ChIP signal in relation to the input signal at binding sites (see Experimental Procedures for mathematical details) and distributed between −4 and 12 (color map scale).

    Article Snippet: Co-immunoprecipitation was done using standard techniques and anti-CDX2 (BD Pharmingen 560171; Extended Experimental Procedures).

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    An epigenomic screen identifies CDX2 as a candidate regulator of intestinal epithelial maturation A , Caco-2 cells show progenitor properties while proliferating (left); after reaching confluence, the cells differentiate to resemble mature intestinal villus enterocytes (right). Histograms depict EdU incorporation, a marker of DNA replication, 1 h after exposure, as measured by fluorescence intensity. Diagrams in the upper right represent cell morphologies. B , Chromatin modifications associated with nascent (left) and active (right) enhancers are depicted, the latter characterized by relative increase in H3K4Me2 on flanking nucleosomes and depletion of signal in the nucleosome in between. C , H3K4Me2 ChIP-seq results in proliferating and differentiated Caco-2 cells at a representative region on chromosome 10. D , H3K4Me2 ChIP-seq tag counts across indicated 1-kb intervals in proliferating (left, blue) and differentiated (right, red) cells, including high- resolution image of the data shown in C. The top 2 panels show gain of active chromatin structure upon differentiation; the lower panel shows a region selectively active earlier in proliferating cells. E . F , The same analysis as in E, but on the 1,000 regions with the greatest differential chromatin structure in differentiated cells.

    Journal: Developmental cell

    Article Title: Differentiation-specific histone modifications reveal dynamic chromatin interactions and alternative partners for the intestinal transcription factor CDX2

    doi: 10.1016/j.devcel.2010.10.006

    Figure Lengend Snippet: An epigenomic screen identifies CDX2 as a candidate regulator of intestinal epithelial maturation A , Caco-2 cells show progenitor properties while proliferating (left); after reaching confluence, the cells differentiate to resemble mature intestinal villus enterocytes (right). Histograms depict EdU incorporation, a marker of DNA replication, 1 h after exposure, as measured by fluorescence intensity. Diagrams in the upper right represent cell morphologies. B , Chromatin modifications associated with nascent (left) and active (right) enhancers are depicted, the latter characterized by relative increase in H3K4Me2 on flanking nucleosomes and depletion of signal in the nucleosome in between. C , H3K4Me2 ChIP-seq results in proliferating and differentiated Caco-2 cells at a representative region on chromosome 10. D , H3K4Me2 ChIP-seq tag counts across indicated 1-kb intervals in proliferating (left, blue) and differentiated (right, red) cells, including high- resolution image of the data shown in C. The top 2 panels show gain of active chromatin structure upon differentiation; the lower panel shows a region selectively active earlier in proliferating cells. E . F , The same analysis as in E, but on the 1,000 regions with the greatest differential chromatin structure in differentiated cells.

    Article Snippet: Co-immunoprecipitation was done using standard techniques and anti-CDX2 (BD Pharmingen 560171; Extended Experimental Procedures).

    Techniques: Marker, Fluorescence, Chromatin Immunoprecipitation

    Immunostaining of CDX2. (A) IVF-derived hatched blastocysts (100X) and (B) TE cells produced under feeder-free conditions showing positive expression of CDX2 (200X). Scale bar = 100 μm.

    Journal: PLoS ONE

    Article Title: Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

    doi: 10.1371/journal.pone.0129235

    Figure Lengend Snippet: Immunostaining of CDX2. (A) IVF-derived hatched blastocysts (100X) and (B) TE cells produced under feeder-free conditions showing positive expression of CDX2 (200X). Scale bar = 100 μm.

    Article Snippet: After thorough washing with DPBS, the cells were incubated with the blocking solution (5% BSA) for 1h, followed by an overnight incubation at 4°C with the primary antibody which included anti-CDX2 (ready-to-use, AM392-10M, Bio-Genex Inc., San Ramon, CA, USA), anti-cytokeratin-18(1:200, sc-32329, SantaCruz Biotechnology, Dallas, TX, USA), anti-keratin(1:500, MAB1611, Milipore, Temecula, CA, USA), anti-vimentin (1:200, V6630, Sigma), anti-tubulin (1:400,T8328, Sigma).

    Techniques: Immunostaining, Derivative Assay, Produced, Expressing

    Global CDX2 levels in different types of embryos. Global level of CDX2 in cloned embryos produced using trophoblast cells, adult fibroblasts and fetal fibroblasts as donor cells and those produced by IVF. Bars marked with an asterisk differ significantly from corresponding values (P

    Journal: PLoS ONE

    Article Title: Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

    doi: 10.1371/journal.pone.0129235

    Figure Lengend Snippet: Global CDX2 levels in different types of embryos. Global level of CDX2 in cloned embryos produced using trophoblast cells, adult fibroblasts and fetal fibroblasts as donor cells and those produced by IVF. Bars marked with an asterisk differ significantly from corresponding values (P

    Article Snippet: After thorough washing with DPBS, the cells were incubated with the blocking solution (5% BSA) for 1h, followed by an overnight incubation at 4°C with the primary antibody which included anti-CDX2 (ready-to-use, AM392-10M, Bio-Genex Inc., San Ramon, CA, USA), anti-cytokeratin-18(1:200, sc-32329, SantaCruz Biotechnology, Dallas, TX, USA), anti-keratin(1:500, MAB1611, Milipore, Temecula, CA, USA), anti-vimentin (1:200, V6630, Sigma), anti-tubulin (1:400,T8328, Sigma).

    Techniques: Clone Assay, Produced

    Relative mRNA abundance of some important genes in different types of embryos. Expression level of epigenetics- ( DNMT1 and DNMT3a ), trophectoderm- ( IFN-tau , CDX2 , GATA2 and GATA3 ) and pluripotency-related ( OCT4 and SOX2 ) genes in cloned embryos produced using trophoblast cells, adult fibroblasts and fetal fibroblasts as donor cells and those produced by IVF. Bars with different superscripts differ significantly (P

    Journal: PLoS ONE

    Article Title: Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

    doi: 10.1371/journal.pone.0129235

    Figure Lengend Snippet: Relative mRNA abundance of some important genes in different types of embryos. Expression level of epigenetics- ( DNMT1 and DNMT3a ), trophectoderm- ( IFN-tau , CDX2 , GATA2 and GATA3 ) and pluripotency-related ( OCT4 and SOX2 ) genes in cloned embryos produced using trophoblast cells, adult fibroblasts and fetal fibroblasts as donor cells and those produced by IVF. Bars with different superscripts differ significantly (P

    Article Snippet: After thorough washing with DPBS, the cells were incubated with the blocking solution (5% BSA) for 1h, followed by an overnight incubation at 4°C with the primary antibody which included anti-CDX2 (ready-to-use, AM392-10M, Bio-Genex Inc., San Ramon, CA, USA), anti-cytokeratin-18(1:200, sc-32329, SantaCruz Biotechnology, Dallas, TX, USA), anti-keratin(1:500, MAB1611, Milipore, Temecula, CA, USA), anti-vimentin (1:200, V6630, Sigma), anti-tubulin (1:400,T8328, Sigma).

    Techniques: Expressing, Clone Assay, Produced

    (A) Kaplan–Meier survival curve showing effect of Cdx2 expression on overall survival ( n = 93). (B) ROC curve highlighting the strong predictive effect and specificity of loss of Cdx2 expression for MMR-deficiency. (C) Kaplan–Meier survival curve showing effect of Cdx2 expression on overall survival in MMR-proficient colorectal cancer patients only ( n = 78). Wilcoxon’s test.

    Journal: Frontiers in Oncology

    Article Title: Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

    doi: 10.3389/fonc.2013.00265

    Figure Lengend Snippet: (A) Kaplan–Meier survival curve showing effect of Cdx2 expression on overall survival ( n = 93). (B) ROC curve highlighting the strong predictive effect and specificity of loss of Cdx2 expression for MMR-deficiency. (C) Kaplan–Meier survival curve showing effect of Cdx2 expression on overall survival in MMR-proficient colorectal cancer patients only ( n = 78). Wilcoxon’s test.

    Article Snippet: An automated Bond III instrument was used along with the following antibodies and protocols: Cdx2, Leica-Novocastra, NCL-Cdx2, 1:200, Tris 95°, 30 min; MLH1, Leica-Novocastra, NCL-MLH1, 1:200, Tris 95°, 30 min; MSH2, Leica-Novocastra, NCL-MSH2, 1:200, Tris 95°, 30 min; MSH6, Leica-Novocastra, MSH6-L-CE, 1:1600, Tris 95°, 30 min; PMS2, Leica-Novocastra NCL-L-PMS2, 1:75, Tris 95°, 30 min. For Cdx2 expression, the percentage of positive tumor cells was estimated.

    Techniques: Expressing

    (A) Diffuse and (B) focal expression of Cdx2 in colorectal cancers.

    Journal: Frontiers in Oncology

    Article Title: Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

    doi: 10.3389/fonc.2013.00265

    Figure Lengend Snippet: (A) Diffuse and (B) focal expression of Cdx2 in colorectal cancers.

    Article Snippet: An automated Bond III instrument was used along with the following antibodies and protocols: Cdx2, Leica-Novocastra, NCL-Cdx2, 1:200, Tris 95°, 30 min; MLH1, Leica-Novocastra, NCL-MLH1, 1:200, Tris 95°, 30 min; MSH2, Leica-Novocastra, NCL-MSH2, 1:200, Tris 95°, 30 min; MSH6, Leica-Novocastra, MSH6-L-CE, 1:1600, Tris 95°, 30 min; PMS2, Leica-Novocastra NCL-L-PMS2, 1:75, Tris 95°, 30 min. For Cdx2 expression, the percentage of positive tumor cells was estimated.

    Techniques: Expressing

    Schematic diagram illustrating a proposal for the involvement of Cdx2 during progression of cancers through the serrated pathway .

    Journal: Frontiers in Oncology

    Article Title: Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

    doi: 10.3389/fonc.2013.00265

    Figure Lengend Snippet: Schematic diagram illustrating a proposal for the involvement of Cdx2 during progression of cancers through the serrated pathway .

    Article Snippet: An automated Bond III instrument was used along with the following antibodies and protocols: Cdx2, Leica-Novocastra, NCL-Cdx2, 1:200, Tris 95°, 30 min; MLH1, Leica-Novocastra, NCL-MLH1, 1:200, Tris 95°, 30 min; MSH2, Leica-Novocastra, NCL-MSH2, 1:200, Tris 95°, 30 min; MSH6, Leica-Novocastra, MSH6-L-CE, 1:1600, Tris 95°, 30 min; PMS2, Leica-Novocastra NCL-L-PMS2, 1:75, Tris 95°, 30 min. For Cdx2 expression, the percentage of positive tumor cells was estimated.

    Techniques:

    The mean apoptotic rate in MGC-803/Cdx2 small interference RNA cells was significantly higher than that in MGC-803/Cdx2 negative control or MGC-803 cells. Percentages of apoptotic cells analyzed by flow cytometry. Numbers in the quadrants reflected the

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: The mean apoptotic rate in MGC-803/Cdx2 small interference RNA cells was significantly higher than that in MGC-803/Cdx2 negative control or MGC-803 cells. Percentages of apoptotic cells analyzed by flow cytometry. Numbers in the quadrants reflected the

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Negative Control, Flow Cytometry, Cytometry

    Cdx2 small interference RNA upregulated phosphatase and tensin homolog, caspase-9 and caspase-3 mRNA expression. A: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 μg in each) extracted from

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA upregulated phosphatase and tensin homolog, caspase-9 and caspase-3 mRNA expression. A: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 μg in each) extracted from

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Cdx2 mRNA and protein expression was suppressed in MGC-803/Cdx2 small interference RNA tumor tissue. A: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Total RNAs (2 μg in each) extracted from tumor tissue

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 mRNA and protein expression was suppressed in MGC-803/Cdx2 small interference RNA tumor tissue. A: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Total RNAs (2 μg in each) extracted from tumor tissue

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Cdx2 small interference RNA decreased invasion of MGC-803 cells. MGC-803 cells (A), MGC-803/Cdx2 negative control cells (B) and MGC-803/Cdx2 small interference RNA cells (C) were loaded onto Matrigel-coated upper chambers of Transwell plates. Filtrated

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA decreased invasion of MGC-803 cells. MGC-803 cells (A), MGC-803/Cdx2 negative control cells (B) and MGC-803/Cdx2 small interference RNA cells (C) were loaded onto Matrigel-coated upper chambers of Transwell plates. Filtrated

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Negative Control

    Cdx2 small interference RNA significantly increased phosphatase and tensin homolog, cleaved caspase-9 and cleaved caspase-3 protein concentrations while pro-caspase-9 and pro-caspase-3 are decreased. A: Western blotting analysis. Whole protein extracts

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA significantly increased phosphatase and tensin homolog, cleaved caspase-9 and cleaved caspase-3 protein concentrations while pro-caspase-9 and pro-caspase-3 are decreased. A: Western blotting analysis. Whole protein extracts

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Western Blot

    Cdx2 small interference RNA promoted tumor cells apoptosis. Tumor cells were assessed for apoptosis using terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine, 5’-triphosphate nick end labeling assay. The apoptotic cells were

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA promoted tumor cells apoptosis. Tumor cells were assessed for apoptosis using terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine, 5’-triphosphate nick end labeling assay. The apoptotic cells were

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: End Labeling

    Cdx2 small interference RNA inhibits cell proliferation in MGC-803 cells. MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA inhibits cell proliferation in MGC-803 cells. MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Negative Control

    MGC-803/Cdx2 small interference RNA cells exhibited fewer colonies than MGC-803/Cdx2 negative control cells or MGC-803 cells. A: MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells were plated in 6-well

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: MGC-803/Cdx2 small interference RNA cells exhibited fewer colonies than MGC-803/Cdx2 negative control cells or MGC-803 cells. A: MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells were plated in 6-well

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Negative Control

    Cdx2 small interference RNA caused cell cycle arrest in the G0/G1 phase. Cell cycle was analyzed by flow cytometry in MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells. The data were representative

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA caused cell cycle arrest in the G0/G1 phase. Cell cycle was analyzed by flow cytometry in MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells. The data were representative

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Flow Cytometry, Cytometry, Negative Control

    Cdx2 small interference RNA decreased migration of MGC-803 cells in wound healing assay. MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells were cultured to confluence on 6-well plates, a central linear

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA decreased migration of MGC-803 cells in wound healing assay. MGC-803 cells, MGC-803/Cdx2 negative control cells and MGC-803/Cdx2 small interference RNA (siRNA) cells were cultured to confluence on 6-well plates, a central linear

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Migration, Wound Healing Assay, Negative Control, Cell Culture

    Cdx2 small interference RNA significantly reduced Cdx2 mRNA and protein expression in MGC-803 cells. A: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 μg in each) extracted from MGC-803

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Cdx2 small interference RNA significantly reduced Cdx2 mRNA and protein expression in MGC-803 cells. A: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 μg in each) extracted from MGC-803

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Tumor growth curve showed a significant growth tendency in MGC-803 cells and in MGC-803/Cdx2 negative control cells, while the tumor growth in MGC-803/Cdx2 small interference RNA cells was obviously inhibited. MGC-803 cells, MGC-803/Cdx2 negative control

    Journal: World Journal of Gastroenterology : WJG

    Article Title: siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

    doi: 10.3748/wjg.v18.i16.1903

    Figure Lengend Snippet: Tumor growth curve showed a significant growth tendency in MGC-803 cells and in MGC-803/Cdx2 negative control cells, while the tumor growth in MGC-803/Cdx2 small interference RNA cells was obviously inhibited. MGC-803 cells, MGC-803/Cdx2 negative control

    Article Snippet: Anti-Cdx2, anti-β-actin and secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States.

    Techniques: Negative Control

    Upper panel: qPCR analysis of CDX2-negative HT29 cells treated with increasing concentrations of the DNMTi decitabine (1.25 μM, 2.5 μM, 5 μM, 10 μM) for 48 h and increasing concentrations of the HDAC4/5i LMK-235 (5 nM, 10 nM, 20 nM, 40 nM, 80 nM). Data were normalized to the HMBS housekeeping gene and are shown as n -fold regulation compared with DMSO-treated cells. MWU: *** p

    Journal: Clinical Epigenetics

    Article Title: CDX2 in colorectal cancer is an independent prognostic factor and regulated by promoter methylation and histone deacetylation in tumors of the serrated pathway

    doi: 10.1186/s13148-018-0548-2

    Figure Lengend Snippet: Upper panel: qPCR analysis of CDX2-negative HT29 cells treated with increasing concentrations of the DNMTi decitabine (1.25 μM, 2.5 μM, 5 μM, 10 μM) for 48 h and increasing concentrations of the HDAC4/5i LMK-235 (5 nM, 10 nM, 20 nM, 40 nM, 80 nM). Data were normalized to the HMBS housekeeping gene and are shown as n -fold regulation compared with DMSO-treated cells. MWU: *** p

    Article Snippet: Membranes were blocked with 5% TBS-milk for at least 45 min. Blots were incubated with anti-CDX2 (1:500 in BSA, EPR2764Y—28.8 μg/mL, Rabbit Monoclonal, Cell Marque; Sigma-Aldrich) over night at 4 °C followed by incubation with anti-rabbit (1:10,000 in milk; Cell Signalling Technology, Leiden, The Netherlands) for at least 2 h at RT.

    Techniques: Real-time Polymerase Chain Reaction

    HDAC5 regulates CDX2 expression by binding to the promoter region of the CDX2 gene. a Schematic representation of the genomic region upstream of the transcriptional start site (TSS) of the CDX2 gene. ChIP primer locations are indicated by arrows. b In vivo binding of HDAC5 to the indicated genomic regions upstream of the CDX2 TSS was shown by ChIP in 293T cells transfected with FLAG-tagged HDAC5 using antibodies against FLAG. Antibodies against acetyl-histone H3 and mouse IgG were used as positive and negative controls, respectively. Amplification of a genomic region in the FGF2 gene was shown as a positive control for a genomic region bound by HDAC5. *unspecific PCR band

    Journal: Clinical Epigenetics

    Article Title: CDX2 in colorectal cancer is an independent prognostic factor and regulated by promoter methylation and histone deacetylation in tumors of the serrated pathway

    doi: 10.1186/s13148-018-0548-2

    Figure Lengend Snippet: HDAC5 regulates CDX2 expression by binding to the promoter region of the CDX2 gene. a Schematic representation of the genomic region upstream of the transcriptional start site (TSS) of the CDX2 gene. ChIP primer locations are indicated by arrows. b In vivo binding of HDAC5 to the indicated genomic regions upstream of the CDX2 TSS was shown by ChIP in 293T cells transfected with FLAG-tagged HDAC5 using antibodies against FLAG. Antibodies against acetyl-histone H3 and mouse IgG were used as positive and negative controls, respectively. Amplification of a genomic region in the FGF2 gene was shown as a positive control for a genomic region bound by HDAC5. *unspecific PCR band

    Article Snippet: Membranes were blocked with 5% TBS-milk for at least 45 min. Blots were incubated with anti-CDX2 (1:500 in BSA, EPR2764Y—28.8 μg/mL, Rabbit Monoclonal, Cell Marque; Sigma-Aldrich) over night at 4 °C followed by incubation with anti-rabbit (1:10,000 in milk; Cell Signalling Technology, Leiden, The Netherlands) for at least 2 h at RT.

    Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, In Vivo, Transfection, Amplification, Positive Control, Polymerase Chain Reaction

    Eight colorectal cancer cell lines showing the expected inverse correlation between hypermethylation percentage at two CDX2 promoter sequences and CDX2 protein expression

    Journal: Clinical Epigenetics

    Article Title: CDX2 in colorectal cancer is an independent prognostic factor and regulated by promoter methylation and histone deacetylation in tumors of the serrated pathway

    doi: 10.1186/s13148-018-0548-2

    Figure Lengend Snippet: Eight colorectal cancer cell lines showing the expected inverse correlation between hypermethylation percentage at two CDX2 promoter sequences and CDX2 protein expression

    Article Snippet: Membranes were blocked with 5% TBS-milk for at least 45 min. Blots were incubated with anti-CDX2 (1:500 in BSA, EPR2764Y—28.8 μg/mL, Rabbit Monoclonal, Cell Marque; Sigma-Aldrich) over night at 4 °C followed by incubation with anti-rabbit (1:10,000 in milk; Cell Signalling Technology, Leiden, The Netherlands) for at least 2 h at RT.

    Techniques: Expressing

    Decitabine significantly restores CDX2 expression in CDX2-negative CRC cell lines. a Upper panel: qPCR analysis of CDX2-negative COLO205 and SW620 cells treated with increasing concentrations of the DNMTi decitabine (1.25 μM, 2.5 μM, 5 μM) for 48 h. Data were normalized to the HMBS housekeeping gene and are shown as n -fold regulation compared with DMSO-treated cells. MWU: *** p

    Journal: Clinical Epigenetics

    Article Title: CDX2 in colorectal cancer is an independent prognostic factor and regulated by promoter methylation and histone deacetylation in tumors of the serrated pathway

    doi: 10.1186/s13148-018-0548-2

    Figure Lengend Snippet: Decitabine significantly restores CDX2 expression in CDX2-negative CRC cell lines. a Upper panel: qPCR analysis of CDX2-negative COLO205 and SW620 cells treated with increasing concentrations of the DNMTi decitabine (1.25 μM, 2.5 μM, 5 μM) for 48 h. Data were normalized to the HMBS housekeeping gene and are shown as n -fold regulation compared with DMSO-treated cells. MWU: *** p

    Article Snippet: Membranes were blocked with 5% TBS-milk for at least 45 min. Blots were incubated with anti-CDX2 (1:500 in BSA, EPR2764Y—28.8 μg/mL, Rabbit Monoclonal, Cell Marque; Sigma-Aldrich) over night at 4 °C followed by incubation with anti-rabbit (1:10,000 in milk; Cell Signalling Technology, Leiden, The Netherlands) for at least 2 h at RT.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Immunohistochemical (immunoperoxidase and hematoxylin counterstain) findings of synchronous primary carcinoid tumor and primary adenocarcinoma arising within mature cystic teratoma of horseshoe kidney . (A) CDX2 staining was positive in the mucinous columnar enteric-type or colonic-like epithelium lining of teratomatous cysts. Original magnification ×200. (B) Cytokeratin 20 staining was positive in the mucinous columnar enteric-type or colonic-like epithelium lining of teratomatous cysts. Original magnification ×200. (C) Smooth muscle actin was positive in teratomatous cyst wall. Original magnification ×200. (D) Cytokeratin 7 staining was positive in the adenocarcinoma component in right upper part of figure, but negative in the carcinoid tumor component in left lower part of figure. Original magnification ×200.

    Journal: Diagnostic Pathology

    Article Title: Synchronous primary carcinoid tumor and primary adenocarcinoma arising within mature cystic teratoma of horseshoe kidney: a unique case report and review of the literature

    doi: 10.1186/1746-1596-4-17

    Figure Lengend Snippet: Immunohistochemical (immunoperoxidase and hematoxylin counterstain) findings of synchronous primary carcinoid tumor and primary adenocarcinoma arising within mature cystic teratoma of horseshoe kidney . (A) CDX2 staining was positive in the mucinous columnar enteric-type or colonic-like epithelium lining of teratomatous cysts. Original magnification ×200. (B) Cytokeratin 20 staining was positive in the mucinous columnar enteric-type or colonic-like epithelium lining of teratomatous cysts. Original magnification ×200. (C) Smooth muscle actin was positive in teratomatous cyst wall. Original magnification ×200. (D) Cytokeratin 7 staining was positive in the adenocarcinoma component in right upper part of figure, but negative in the carcinoid tumor component in left lower part of figure. Original magnification ×200.

    Article Snippet: The antibodies used in this study included the following; alpha-methylacyl-coenzyme A-racemase (AMACR/P504S) [1:100, Ventana Medical Systems Inc, Tucson, Arizona], calretinin (1:100, Becton Dickinson, San Jose, California, USA), carbonic anhydrase IX (CA-IX, 1:200, DakoCytomation, Carpinteria, California, USA), CD10 (1:200, Ventana), CD56 (1:200, DakoCytomation), CD99 (1:100, Ventana), CDX2 (1:100, Ventana), chromogranin (1:200, Signet Pathology Systems Inc, Dedham, Massachusetts, USA), cytokeratin 7 (CK7, 1:200, Ventana), cytokeratin 20 (CK20, 1:200, DakoCytomation), pancytokeratin cocktail (AE1-AE3, 1:500, DakoCytomation; CAM 5.2, 1:50, Becton Dickinson; MNF116, 1:50, DakoCytomation; and UCD/PR-10.11, 1:25, Zymed, San Francisco, California), smooth muscle actin (SMA, 1:200, DakoCytomation), synaptophysin (1:100, DakoCytomation), and thyroid transcription factor-1 (TTF-1, 1:250, Ventana).

    Techniques: Immunohistochemistry, Staining

    Depletion of architectural proteins alters histone modifications and transcription factor occupancy. ( A )–( C ) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t -test, P -values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. ( D )–( F ) ChIP for FOXA1 (D), FOXA2 (E) and CDX2 (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. * P

    Journal: Nucleic Acids Research

    Article Title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

    doi: 10.1093/nar/gku648

    Figure Lengend Snippet: Depletion of architectural proteins alters histone modifications and transcription factor occupancy. ( A )–( C ) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t -test, P -values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. ( D )–( F ) ChIP for FOXA1 (D), FOXA2 (E) and CDX2 (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. * P

    Article Snippet: Antibodies were against FOXA1 (Abcam ab5089), CDX2 (Bethyl Laboratories A300–691A), FOXA2 (SCB sc-6554x), histone H3 (ab1791), H3K9Me3 (ab8898), H3K9Ac (Millipore 07–352), H3K27ac (ab4729), CTCF (Millipore 07–729), RAD21 (ab992), SMC1 (Bethyl Laboratories A300–055A), normal goat IgG (SCB sc-2028), or normal rabbit IgG (Millipore 12–370). qPCR was performed using SYBR® Green reagents with primers listed in Supplementary Table S1.

    Techniques: Chromatin Immunoprecipitation, Transfection, Modification, Two Tailed Test