Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 1. Genomic organization of the human ELF5 locus and transcript isoform expression in placenta and trophoblast cell lines. (A) Diagram of the exon–intron structure of the human ELF5 locus and annotated splice var- iants. Position of primers used is indicated. Filled boxes represent open- reading frames and open boxes represent untranslated regions. (B) RT–PCR analysis with isoform-specific and common primers reveals that ELF5-2b is the expressed splice variant in placenta and the trophoblast-like cell line TCL-1, but that it is absent from the first trimester mesenchymal-like cell line TCL-2. (C) RT–PCR with primers spanning exons 3 and 4 demonstrates that the annotated ELF5-2bDex3/4 variant is not present in placenta and chor- iocarcinoma and trophoblast-like cell lines JEG-3 and TCL-1.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 2. Expression of trophoblast stem cell genes and epigenetic regulation of ELF5 in placenta throughout gestation. (A) RT–PCR analysis of ELF5, CDX2 and EOMES (i.e. genes important for trophoblast stem cell self-renewal and proliferation in the mouse) on human placental villous samples ranging from 7 weeks of gestation to term. Four independent term placental samples were investigated. The choriocarcinoma cell line JEG-3 was included as control. Colour-inverted photographs of ethidium bromide stained gels are shown. All three genes are expressed in placenta, but CDX2 is not detected from the second trimester onwards even when the PCRs are over-cycled. (B) Quantitative RT–PCR (qPCR) analysis of ELF5, CDX2 and EOMES on the same samples used in (A). ELF5 is down- regulated in second and third trimesters, whereas no overall regulation with gestational age was observed for EOMES. (C) Comparison of expression levels between first trimester and term. ELF5 expression is significantly reduced at term when compared with first trimester, CDX2 is absent from term placentas. (D) Bisulphite sequencing analysis of the ELF5 promoter region. Filled circles indicate methylated cytosine residues. ELF5 is extremely hypomethylated in the first trimester and acquires higher DNA methylation levels in second and third trimester, correlating with transcriptional down-regulation at these stages. (E) DNA methylation analysis of an extended region between 2400 bp and +400 bp around the transcriptional start site of ELF5. Hypomethylation correlates with ELF5 expression in JEG-3 cells and, conversely, ELF5 is hypermethylated and not expressed in TCL-2 cells. The methylation pattern in TCL-1 cells reveals a critical stretch of five CpG residues (grey box) at the immediate transcriptional start site that needs to be unmethylated for ELF5 to be expressed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Quantitative RT-PCR, Comparison, Bisulfite Sequencing, Methylation, DNA Methylation Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 3. Immunofluorescence localization of ELF5 to cytotrophoblasts in the human placenta. (A) Overview of 11 week placental villous cross-section shows ELF5 localization to nuclei of villous cytotrophoblasts, but absence from nuclei of the overlying syncytiotrophoblast layer. Cytotrophoblasts are a proliferative cell population that continuously divide to replenish the overlying syncytium. (B) Co-localization with cytokeratin 7 (CK7) confirms the trophoblast identity of ELF5-positive cells. (C) Confocal image of a double staining of ELF5 and the villous cytotrophoblast marker SPINT1 (also known as HAI-1) shows that every ELF5-positive nucleus resides within the cytotrophoblast layer. Top row 6 week, bottom row 11 week placenta. (D) Confocal image analysis of an 11 week villous section stained for ELF5 and the extravillous cytotrophoblast (EVT) marker integrin alpha-5 (ITGA5). ELF5 is detected only in nuclei at the proliferative base, but not further distal along the EVT column where cells adopt an invasive phenotype and lose proliferative potential. (E) ELF5 is also absent from post- mitotic interstitial and endovascular EVTs within the decidual bed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Double Staining, Marker, Staining

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 4. CDX2 identifies a subset of ELF5-positive cytotrophoblasts as a TS-like compartment that is regulated by FGFR2. (A) ELF5 co-localizes with FGFR2 in villous cytotrophoblasts as identified by confocal image analysis of double immunofluorescence stainings of 11 week placental sections. Since FGF signalling has been implicated in TS cell proliferation in mice and humans and can activate ELF5 expression in other tissues, FGF/FGFR2 may induce ELF5 expression within a putative TS cell niche in the human placenta. (B) Double staining of a 6 week placental section for ELF5 and CDX2. Larger groups of CDX2-positive cells are detected only in early gestation up to 8.5–9 weeks. CDX2 is mostly co-expressed with ELF5 (arrowheads). (C) Dual labelling of 6 week placental section for CDX2 and the proliferation marker Ki67. CDX2-expressing cytotrophoblasts preferentially stain positive for Ki67, indicating their high proliferation rate. CDX2 and Ki67 are restricted to the proximal end of cytotrophoblast cell columns (highlighted by the boxed area). The white arrows indicate the direction of progressive extravillous trophoblast (EVT) differentiation and migration.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Double Staining, Marker, Staining, Migration

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 5. Inter-regulatory network of trophoblast transcription factors CDX2, EOMES and ELF5. (A) Chromatin immunoprecipitation assays show that CDX2 binds to the ELF5 promoter region in JEG-3 and TCL-1 cells where ELF5 is hypomethylated and expressed, but not in TCL-2 cells where ELF5 is hypermethylated and repressed. (B) In turn, ELF5 binds to the CDX2 and EOMES promoter regions in JEG-3 and TCL-1 cells where it is expressed, but not in TCL-2 cells from which it is absent, thereby establishing a transcrip- tional feedback loop between all three transcription factors. Binding to the EOMES promoter region was more consistent and is indicative of a more effi- cient, stronger interaction than with the CDX2 upstream region, consistent with results observed in mouse trophoblast (9).

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Chromatin Immunoprecipitation, Binding Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 6. Trophoblast transcription factor expression and epigenetic regulation of ELF5 in human ES cells and derived trophoblast cell lines. (A) Initial bisul- phite sequencing analysis of two pooled hES cell lines and derived trophoblast cells indicates a high degree of DNA methylation at the ELF5 promoter despite the limited trophoblast differentiation potential. (B) RT–PCR and (C) qPCR analysis for trophoblast transcription factors ELF5, CDX2 and EOMES on six differ- ent hES cells lines (Shef1, Shef4–7, H7), including one subclone with an abnormal karyotype (Shef5a), two derived cytotrophoblast cell lines (TrophH7 and TrophShef4), the JEG-3, TCL-1 and TCL-2 cell lines, an 8+4 week placenta for relative comparison of expression levels and a colorectal cancer cell line (DKO4) as positive control for CDX2 expression (27). Colour-inverted photographs of ethidium bromide stained gels are shown. ELF5 is detectable in some hES cell lines, albeit at very low levels. Higher expression levels of CDX2 and EOMES may relate to their function within the embryonic lineage and is not directly indicative of trophoblast differentiation potential. Strikingly, in contrast to their expression in placenta, all three genes are absent from the hES-derived tropho- blast cell lines. (D) Normalization of qPCR data to Shef6, one of the most highly ELF5 expressing hES cell lines, in comparison with JEG-3, TCL-1 and TCL-2 cell lines as well as a first trimester placenta sample demonstrates the comparatively negligible amount of ELF5 expression in hES cells that is approximately 300-fold less than in normal trophoblast in vivo. (E) Bisulphite sequencing analysis of the ELF5 promoter in three different hES cell lines and two derived trophoblast cell lines shows relatively little epigenetic variability between different hES cell lines. Hypermethylation correlates with extremely low ELF5 expression levels. (F) Elf5 is also highly methylated in three independent mouse epiblast stem cell lines and (G) in two human-induced pluripotent stem cell lines derived from kereatinocytes and fibroblasts.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Derivative Assay, Sequencing, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Comparison, Positive Control, Staining, In Vivo, Bisulfite Sequencing, Methylation

Journal: The International journal of developmental biology

Article Title: Chromatin organization and timing of polar body I extrusion identify developmentally competent mouse oocytes.

doi: 10.1387/ijdb.180362sg

Figure Lengend Snippet: Fig. 4. Immuno-detection of CDX2- and OCT4-positive blastomeres in blastocysts. Blastocysts developed from fertilized group A or ovulated MII oocytes. Blastocysts were counterstained with DAPI used to count the total number of blastomeres. The number of blastomeres was not significantly different (p> 0.05) among the two groups analyzed. Bar, 10 mm.

Article Snippet: Embryos were then processed for sequential immunolabeling using a rabbit polyclonal anti-human OCT4 (Abcam, cat. n. ab19857) and a rabbit anti-human CDX2 (Cell Signaling, cat. n 3977S) antibody.

Techniques:

Journal: American Journal of Cancer Research

Article Title: HDAC6/FOXP3/HNF4α axis promotes bile acids induced gastric intestinal metaplasia

doi:

Figure Lengend Snippet: FOXP3 inhibited the expression of intestinal markers in gastric cells. A. Downregulation of FOXP3 caused consequential enhancement of MUC2, KLF4 and CDX2 in GES-1 cells. B. Intestinal markers were decreased by FOXP3 overexpression in AGS cells. C. The enhancement of intestinal markers induced by HDAC6 overexpression was diminished by upregulation of FOXP3 in GES-1 cells. D. Knockdown of FOXP3 rescued expression of intestinal markers reduced by siHDAC6 in AGS cells. **P < 0.01. N.S., not significant.

Article Snippet: The following antibodies were used in this study: anti-HDAC6 (1:1000, Cell Signaling Technology, #7558), anti-FOXP3 (1:2000, abcam, #ab10901), anti-HNF4α (1:2000, abcam, #ab72378), anti-CDX2 (1:1000, Cell Signaling Technology, #12306), anti-KLF4 (1:1000, Cell Signaling Technology, #12173), anti-MUC2 (1:4000, abcam, ab134119) and anti-β-actin (1:5000, Bioworld, #AP0060).

Techniques: Expressing, Over Expression

Journal: American Journal of Cancer Research

Article Title: HDAC6/FOXP3/HNF4α axis promotes bile acids induced gastric intestinal metaplasia

doi:

Figure Lengend Snippet: FOXP3 negatively regulated HNF4α transcription. A. FOXP3 inversely regulated HNF4α at both mRNA and protein levels. Student’s t-test. B. The promoter activity of HNF4α was enhanced by DCA and siFOXP3 treatment in GES-1 cells. Student’s t-test. C. A ChIP assay demonstrated the direct binding of FOXP3 to the HNF4α promoter in GES-1 cells. Student’s t-test. D. AGS cells were co-infected with FOXP3-expressing vectors and HNF4α-expressing vectors. The expression of intestinal markers was detected by immunoblot. E. Upregulated intestinal markers expression induced by siFOXP3 was attenuated by siHNF4α in GES-1 cells. F. A schematic model of miR-1/HDAC6/FOXP3/HNF4α pathway in gastric cells. In response to BA, miR-1 inhibition promotes HNF4α and HDAC6, which increase the transcription of intestinal markers including MUC2, KLF4 and CDX2. **P < 0.01. N.S., not significant.

Article Snippet: The following antibodies were used in this study: anti-HDAC6 (1:1000, Cell Signaling Technology, #7558), anti-FOXP3 (1:2000, abcam, #ab10901), anti-HNF4α (1:2000, abcam, #ab72378), anti-CDX2 (1:1000, Cell Signaling Technology, #12306), anti-KLF4 (1:1000, Cell Signaling Technology, #12173), anti-MUC2 (1:4000, abcam, ab134119) and anti-β-actin (1:5000, Bioworld, #AP0060).

Techniques: Activity Assay, Binding Assay, Infection, Expressing, Western Blot, Inhibition

Journal: BMC Cancer

Article Title: Impact of CDX2 expression status on the survival of patients after curative resection for colorectal cancer liver metastasis

doi: 10.1186/s12885-018-4902-8

Figure Lengend Snippet: Representatives of CDX2-low and CDX2-high colorectal cancer (CRC). a and b CDX2-low CRC. c and d CDX2-high CRC. Left images a and c : Hematoxylin and eosin staining. Right image b and d : CDX2 immunohistochemistry. Scale bar = 100 μm

Article Snippet: A mouse antihuman CDX2 monoclonal antibody (clone DAK-CDX2, 1:100 dilution, DAKO, Carpinteria, CA, USA) was used as the primary antibody.

Techniques: Staining, Immunohistochemistry

Journal: BMC Cancer

Article Title: Impact of CDX2 expression status on the survival of patients after curative resection for colorectal cancer liver metastasis

doi: 10.1186/s12885-018-4902-8

Figure Lengend Snippet: Clinicopathological characteristics between CDX2-high and CDX2-low colorectal cancer

Article Snippet: A mouse antihuman CDX2 monoclonal antibody (clone DAK-CDX2, 1:100 dilution, DAKO, Carpinteria, CA, USA) was used as the primary antibody.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Impact of CDX2 expression status on the survival of patients after curative resection for colorectal cancer liver metastasis

doi: 10.1186/s12885-018-4902-8

Figure Lengend Snippet: Relationship between CDX2 expression and survival in patients after potentially curative liver metastasectomy

Article Snippet: A mouse antihuman CDX2 monoclonal antibody (clone DAK-CDX2, 1:100 dilution, DAKO, Carpinteria, CA, USA) was used as the primary antibody.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Impact of CDX2 expression status on the survival of patients after curative resection for colorectal cancer liver metastasis

doi: 10.1186/s12885-018-4902-8

Figure Lengend Snippet: Kaplan–Meier curves of disease-free survival (DFS) and overall survival (OS). a The association of CDX2 expression with DFS in patients undergoing potentially curative liver metastasectomy. b The association of CDX2 expression with OS in patients undergoing potentially curative liver metastasectomy. Blue and red lines represent CDX2-low and CDX2-high CRC, respectively

Article Snippet: A mouse antihuman CDX2 monoclonal antibody (clone DAK-CDX2, 1:100 dilution, DAKO, Carpinteria, CA, USA) was used as the primary antibody.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Impact of CDX2 expression status on the survival of patients after curative resection for colorectal cancer liver metastasis

doi: 10.1186/s12885-018-4902-8

Figure Lengend Snippet: Multivariate model to predict DFS and OS in patients after potentially curative liver metastasectomy

Article Snippet: A mouse antihuman CDX2 monoclonal antibody (clone DAK-CDX2, 1:100 dilution, DAKO, Carpinteria, CA, USA) was used as the primary antibody.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Impact of CDX2 expression status on the survival of patients after curative resection for colorectal cancer liver metastasis

doi: 10.1186/s12885-018-4902-8

Figure Lengend Snippet: Kaplan–Meier curves of disease-free survival (DFS) and overall survival (OS) in patients without and with pre- or post-operative chemotherapy. a and b The association of pre- or post-operative chemotherapy with DFS and OS in patients with CDX2-low CRC. c and d The association of pre- or post-operative chemotherapy with DFS and OS in patients with CDX2-high CRC. Red and blue lines represent patients with and without pre- or post-operative chemotherapy, respectively

Article Snippet: A mouse antihuman CDX2 monoclonal antibody (clone DAK-CDX2, 1:100 dilution, DAKO, Carpinteria, CA, USA) was used as the primary antibody.

Techniques:

Journal: BMC Cancer

Article Title: Impact of CDX2 expression status on the survival of patients after curative resection for colorectal cancer liver metastasis

doi: 10.1186/s12885-018-4902-8

Figure Lengend Snippet: Number of sites of recurrence in patients with CDX2-high and CDX2-low colorectal cancer after potentially curative liver metastasectomy

Article Snippet: A mouse antihuman CDX2 monoclonal antibody (clone DAK-CDX2, 1:100 dilution, DAKO, Carpinteria, CA, USA) was used as the primary antibody.

Techniques: Expressing