Journal: Molecular Endocrinology

Article Title: COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

doi: 10.1210/me.2013-1191

Figure Lengend Snippet: Spatiotemporal expression of COUP-TFII in the endometrium of healthy women during the menstrual cycle. A, Immunohistochemical staining of COUP-TFII in zona functionalis at the proliferative phase (a) and the early secretory phase (b), and in zona basalis at the proliferative phase (c) and the midsecretory phase (d) of the menstrual cycle. B, The HSCORE of COUP-TFII in zona functionalis (F) and zona basalis (B) during the menstrual cycle. Proli, proliferative phase; ES, early secretory phase; MS, midsecretory phase; LS, late secretory phase; F, zona functionalis; B, zona basalis. *, P < .05.

Article Snippet: Sections were immune blocked with 10% normal goat serum in PBS and then incubated at 4°C overnight with 1:500 anti-COUP-TFII antibody (PP-H7147–10, Perseus Proteomics, Inc.) in 10% normal goat serum in PBS.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Molecular Endocrinology

Article Title: COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

doi: 10.1210/me.2013-1191

Figure Lengend Snippet: Comparison of COUP-TFII expression in the endometrium between women with and without diagnosed endometriosis. A, qPCR analysis of COUP-TFII gene expression in eutopic endometrium between normal women and the patients with endometriosis during the menstrual cycle normalized using the ddCt method to the 18S gene. B, Representative immunofluorescence staining of COUP-TFII in paired (a and d from patient 1; b and e from patient 2; c and f from patient 3) ectopic and eutopic endometrium. C, Quantification of the immunofluorescence signal of COUP-TFII between eutopic and ectopic endometrium of 9 patients with endometriosis. **, P < .01.

Article Snippet: Sections were immune blocked with 10% normal goat serum in PBS and then incubated at 4°C overnight with 1:500 anti-COUP-TFII antibody (PP-H7147–10, Perseus Proteomics, Inc.) in 10% normal goat serum in PBS.

Techniques: Comparison, Expressing, Gene Expression, Immunofluorescence, Staining

Journal: Molecular Endocrinology

Article Title: COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

doi: 10.1210/me.2013-1191

Figure Lengend Snippet: Gene expression of decidual genes regulated by COUP-TFII knockdown in HESCs at 0, 3, and 6 days after treatment with deciduogenic hormones. A, Gene expression levels of COUP-TFII, IGFBP1, PRL, and WNT4 between COUP-TFII siRNA (siCII) and nontargeting siRNA (siNT) as assessed by qPCR. Gene expression was normalized using the ddCt method to the 18S gene. B, Protein levels of COUP-TFII between siCII and siNT normalized to β-actin as determined by Western blotting. *, P < .05; **, P < .01.

Article Snippet: Sections were immune blocked with 10% normal goat serum in PBS and then incubated at 4°C overnight with 1:500 anti-COUP-TFII antibody (PP-H7147–10, Perseus Proteomics, Inc.) in 10% normal goat serum in PBS.

Techniques: Gene Expression, Knockdown, Western Blot

Journal: Molecular Endocrinology

Article Title: COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

doi: 10.1210/me.2013-1191

Figure Lengend Snippet: Signaling Pathways of Genes Affected by COUP-TFII siRNA Identified by Microarray

Article Snippet: Sections were immune blocked with 10% normal goat serum in PBS and then incubated at 4°C overnight with 1:500 anti-COUP-TFII antibody (PP-H7147–10, Perseus Proteomics, Inc.) in 10% normal goat serum in PBS.

Techniques: Protein-Protein interactions

Journal: Molecular Endocrinology

Article Title: COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

doi: 10.1210/me.2013-1191

Figure Lengend Snippet: Validation of microarray results with qPCR, ELISA, and Western blotting after 3 days of treatment with deciduogenic hormones. A, Fold change in mRNA abundance in COUP-TFII siRNA relative to nontargeting siRNA in HESCs. B and C, Secreted protein levels of IL-6 and IL-8 in HESC culture media. Forty-eight hours after transfection (defined as day 0) with nontargeting siRNA (siNT) or COUP-TFII siRNA (siCII), HESCs were treated with deciduogenic hormones for 3 days. The culture media were collected on day 3, and the levels of IL-6 and IL-8 were measured by ELISA. D, Protein levels of COUP-TFII and PGR isoforms on day 0 and day 3 of treatment with deciduogenic hormones between COUP-TFII siRNA (siCII) and nontargeting siRNA (siNT) normalized to glyceraldehyde-3-phospate dehydrogenase (GAPDH). *, P < .05; **, P < .01.

Article Snippet: Sections were immune blocked with 10% normal goat serum in PBS and then incubated at 4°C overnight with 1:500 anti-COUP-TFII antibody (PP-H7147–10, Perseus Proteomics, Inc.) in 10% normal goat serum in PBS.

Techniques: Biomarker Discovery, Microarray, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection

Journal: Molecular Endocrinology

Article Title: COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

doi: 10.1210/me.2013-1191

Figure Lengend Snippet: ChIP-seq analysis of COUP-TFII in decidualized HESCs. A, Sequence conservation analysis. COUP-TFII ChIP intervals were aligned at the center and expanded 3000 bp around the center. Phastcons scores were retrieved and averaged at each position. B, Distribution of COUP-TFII binding sites throughout the HESC genome. The CEAS module at Cistrome was used for analysis, and the promoter region was defined in increments of 2500 bp to 10 000 bp. The binding locations relative to gene boundaries were further defined as within the 5′-untranslated region (UTR), 3′-UTR, coding exon, or intron. Intergenic regions are defined as more than 10 kb from gene boundaries. C, Frequency of COUP-TFII binding sites located within −10 kb of the transcriptional start site. D, COUP-TFII DNA binding motif enrichment in immunoprecipitated chromatin as determined using the SeqPos tool at Cistrome. E, Venn diagram illustrating the overlap between nonredundant genes bound by COUP-TFII as determined by ChIP-seq and those genes regulated by COUP-TFII by microarray (>1.4-fold). F, UCSC Genome Browser illustrations of select genes with COUP-TFII binding sites. Yellow bars indicate the location of COUP-TFII binding intervals to genes (blue). G, Validation of COUP-TFII binding on the IL8, IL6ST, PGR, IGFBP1, and WNT4 genes by ChIP-qPCR. The putative binding regions identified by ChIP-seq were validated with the COUP-TFII antibody (Active Motif) on HESCs. Antibody against host IgG was used as an immunoprecipitation control and untranslated region (UNTR) primers designed for a region of the human genome devoid of any known genes were used as a qPCR control. Data are represented as signal relative to input. Illustrated statistical significance represents differences in signal between putative binding regions and the UNTR in COUP-TFII-immunoprecipitated chromatin samples. *, P < .05; **, P < .01.

Article Snippet: Sections were immune blocked with 10% normal goat serum in PBS and then incubated at 4°C overnight with 1:500 anti-COUP-TFII antibody (PP-H7147–10, Perseus Proteomics, Inc.) in 10% normal goat serum in PBS.

Techniques: ChIP-sequencing, Sequencing, Binding Assay, Immunoprecipitation, Microarray, Biomarker Discovery, ChIP-qPCR, Control

Journal: Molecular Endocrinology

Article Title: COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

doi: 10.1210/me.2013-1191

Figure Lengend Snippet: Signaling Pathways of Genes With Altered Expression by Microarray That Exhibit COUP-TFII Binding as Identified by ChIP-seq

Article Snippet: Sections were immune blocked with 10% normal goat serum in PBS and then incubated at 4°C overnight with 1:500 anti-COUP-TFII antibody (PP-H7147–10, Perseus Proteomics, Inc.) in 10% normal goat serum in PBS.

Techniques: Protein-Protein interactions, Expressing, Microarray, Binding Assay