Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 1. Mutant C1413S COUP-TFI Dimerizes with wtCOUP-TFI and Inhibits Its DNA Binding Activity A, mutCOUP-TFI interacts with wtCOUP-TFI in a yeast two-hybrid system. Y190 yeast cells were transformed with the different vectors as shown. The resulting b-galactosidase activities were assayed and are shown as Miller units. Each bar represent the mean 6 SEM of four values obtained in two independent experiments. B, mutCOUP-TFI is retained on a GST-wtCOUP-TFI matrix. 4 ml of 35S-labeled in vitro translated mutCOUP-TFI were allowed to interact either with GST alone (lane 2) or a GST-DCOUP-TFI

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Mutagenesis, Binding Assay, Activity Assay, Transformation Assay, Labeling, In Vitro

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 2. Stable Expression of COUP-TFI in Transfected P19 EC Cells A, RT-PCR analysis of transgene expression. Control cells (pcDNA), wild-type (wt), or mutant (mut) COUP-TF expressing cells were grown for 24 h as a monolayer in the presence or absence of cAMP as indicated before RNA extraction and RT-PCR analysis of COUP-TFI and the invariant PO gene expression. Results show the ethidium bromide staining of a 2% agarose gel. B and C, EMSAs of control (pcDNA) and wtCOUP-TFI cells treated as aggregates for 48 h with cAMP. An asterisk indicates the putative Ear2/DNA complex (see text), an arrow shows the position of the COUP-TFI/DNA complex, and an open circle shows the position of the supershifted complex in the presence of COUP-TFI antibodies (Ab). The oligonucleotides indicated in panel C were added as competitors at a 20-fold molar excess. D, DR-1 binding activity in P19 cell aggregates treated with 1026 M RA for 48 h. E, DR-1 binding activities in the different cell lines cultured as aggregates for 48 h and treated as indicated with all-trans-retinoic acid (RA) and/or cAMP. Symbols are the same as in Fig. 1B. Note that the mutant COUP-TFI inhibits endogenous COUP-TFs binding to the probe.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Mutagenesis, RNA Extraction, Gene Expression, Staining, Agarose Gel Electrophoresis, Binding Assay, Activity Assay, Cell Culture

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 4. RT-PCR Analysis of the Expression of Selected Ret- inoid-Responsive Genes RT-PCR analysis was run with RNA extracted from aggre- gates of pcDNA, wt, and mutant COUP-TFI cells treated as indicated for 48 h. Bmp-4, Bone morphogenetic protein-4; E-cad, E-cadherin.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 5. P19 Cell Endogenous COUP-TFs Control Axogenesis Aggregated cells were plated after a 3-day RA treatment and further cultured for 5 days in the presence or not of 1 mM cAMP before anti-NF200 immunohistochemistry (magnification, 3400). Note the absence of neurites in mutCOUP-TFI cells treated with cAMP, despite the presence of neurofilament (NF200)-positive cell bodies (dark staining). Photographs show details of the different cultures and are representative of the content of the entire plates.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Control, Cell Culture, Immunohistochemistry, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 6. COUP-TFI Promotes Neuronal Migration A, Aggregates of cells treated for 3 days with 1 mM RA were plated onto tissue culture-grade plastic and further cultured for 24 h before the RGDS and SDGRG peptides were added at the indicated concentrations. After an additional day in culture, cells were fixed and stained for the presence of NF200 by immunohistochemistry (magnification, 3220). Neurons and neurites were intensely stained and either spread on the top of astrocytes [wtCOUP-TFI (no peptide)], or packed together [mutCOUP-TFI (no peptide), or wtCOUP-TFI RGDS 0.1 mg/ml]. B, The percentage of aggregates presenting either migrating neurons out of the core of the aggregates or a completely spread organization was determined and is shown as the percentage of aggregates with neuron outgrowth in function of the cell lines, in the absence of peptide (control) or in the presence of SDGRG and RGDS peptides at 0.1 mg/ml. Results are shown as the mean 6 SEM for 150 aggregates of each cell line scored in three independent experiments.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Migration, Cell Culture, Staining, Immunohistochemistry, Control

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 8. COUP-TFI Regulates the Synthesis of Vitronectin A, RT-PCR analysis was run with RNA extracted from aggregates (agg.) or monolayer cell cultures (mon.) of pcDNA, wt, and mutant COUP-TFI cells treated as indicated for 48 h. B, Alignment of the human and mouse proximal promoter regions of the vitronectin gene (black dots indicate identical nucleotides between mouse and human, and dashes represent gaps that were introduced to maximize sequence homology). The conserved nuclear receptor (NR) half-binding sites are boxed as well as putative binding sites for other transcription factors. C, The mouse vitronectin promoter is activated by COUP-TFI in transient transfection assays. P19 cells were cotransfected with a mouse vitronectin promoter fragment (2528/147) linked to a luciferase coding sequence and the indicated amounts of wt or mutCOUP-TFI expression vectors. Results are shown as the mean 6 SEM (n 5 3) of the relative luciferase activities (raw luciferase activities divided by b-galactosidase activities).

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Sequencing, Binding Assay, Transfection, Luciferase, Expressing

Journal: eLife

Article Title: The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders

doi: 10.7554/eLife.41770

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti-Nr2f1 , Cell Signalling , 6364 (RRID: AB_11220432 ) , WB (1:1000).

Techniques: Generated, Recombinant