Journal: Nature genetics

Article Title: Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis.

doi: 10.1038/s41588-023-01424-9

Figure Lengend Snippet: Fig. 4 | Cluster A SNVs impair cRE2-mediated silencing in a reporter expression assay in vivo and reduce NR2F1 binding in vitro. a,b, Schematics for in vivo lacZ reporter assay constructs (a) and hindbrain β-galactosidase expression viewed dorsally through the fourth ventricle (b). In b, midline ovals denote IEE/ FBMN progenitors, triangles denote migrating IEEs and leg-like columns denote migrating FBMNs that are highlighted by black arrows in c, d and g–i. c–i, Selected images of ectopic β-galactosidase in transfected embryos (left) and schema (right): cRE1 alone (c, n = 13), cRE3 alone (d, n = 6) cRE2 alone (e, n = 8), cRE1 with cRE2 (f, n = 10), cRE3 with cRE2 (g, n = 7), cRE1 with cRE2 carrying Cluster A variants (h, n = 13) and cRE1 with cRE2 carrying Cluster B variants (i, n = 8). The asterisk denotes a mutant cluster. Scale bar (c), 500 μm and applies to c–i. Additional images are shown in Extended Data Fig. 3. j, Partial cRE2 sequence, as per Fig. 1. Gray horizontal bars denote overlap with in silico, conserved, transcription- binding consensus sequences from TRANSFAC (indicated by $). The shade of gray

Article Snippet: For the supershift assay, 1 μg of anti-NR2F1 antibody (D4H2 rabbit monoclonal antibody, Cell Signaling, catalog no. 6364; mouse monoclonal antibody, Perseus Proteomics, catalog no. PP-H812400) and respective isotype controls (WNT3A rabbit mono clonal antibody, Cell Signaling, catalog no. 2721; anti-hemagglutinin, immunoglobulin G2a mouse monoclonal antibody, Thermo Fisher Scientific, catalog no. 5B1D10) were added during the preincubation step.

Techniques: Expressing, In Vivo, Binding Assay, In Vitro, Reporter Assay, Construct, Transfection, Mutagenesis, Sequencing, In Silico

Journal: Nature genetics

Article Title: Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis.

doi: 10.1038/s41588-023-01424-9

Figure Lengend Snippet: Fig. 5 | NR2F1 binds cRE2 in E10.5 r4MNs and binding is reduced by Fam5 SNV. a, Schematic representation of single-cell CUT&Tag of E10.5 WT and Fam5snv r4 Isl1+ neurons targeting NR2F1. scATAC-seq, single-cell assay for transposase- accessible chromatin with high-throughput sequencing. b, UMAP embedding of NR2F1 single-cell CUT&Tag experiment for two Fam5SNV/SNV (replicate (Rep) 1 = 2,274 cells, Rep 2 = 2,740 cells) and two WT (Rep 1 = 2,572 cells, Rep 2 = 1,377)

Article Snippet: For the supershift assay, 1 μg of anti-NR2F1 antibody (D4H2 rabbit monoclonal antibody, Cell Signaling, catalog no. 6364; mouse monoclonal antibody, Perseus Proteomics, catalog no. PP-H812400) and respective isotype controls (WNT3A rabbit mono clonal antibody, Cell Signaling, catalog no. 2721; anti-hemagglutinin, immunoglobulin G2a mouse monoclonal antibody, Thermo Fisher Scientific, catalog no. 5B1D10) were added during the preincubation step.

Techniques: Binding Assay, Next-Generation Sequencing

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

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Sequences of primers used in this study

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques:

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modifies the expression of the CXCL12 signaling axis in MCF-7 cells. (A) Characterization of the control and COUP clones. An immunofluorescence cytochemistry assay was used to detect the relative expression of HA/COUP-TFI or COUP-TFI proteins in the control (Cont.) and COUP clones. The cells were fixed and processed for immunofluorescence as described in Methods; the nuclei were stained with DAPI. (B) CXCL12 , CXCR4 , and CXCR7 mRNAs were quantified by a real-time PCR analysis from two independent MCF-7 control and COUP clones. The results were normalized to GAPDH mRNA used as an internal control. The results were expressed as the relative mRNA expression level of CXCL12 , CXCR4 , or CXCR7 . Data are the mean values ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (C) The amount of intracellular HA/COUP-TFI, COUP-TFI, CXCL12, CXCR4, and CXCR7 protein was determined from whole-cell extracts of the different MCF-7 clones and compared to total ERK. A representative western blot is shown. (D) The control and COUP clones were fixed, and an immunofluorescence cytochemistry assay was used to detect the relative expression of CXCL12, CXCR4, and CXCR7 proteins. Staining with DAPI is also presented to visualize the nucleus of the cells.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Control, Clone Assay, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modulates the chromatin structure of the CXCL12 and CXCR4 gene promoters. The FAIRE assay was performed as described in Methods. Real-time PCR was performed to monitor the enrichment of DNA corresponding to the proximal promoter of the CXCL12 (A) , the CXCR4 (B) and the CXCR7 (C) genes relative to the input chromatin from the control (Cont.) and COUP clones. The data are from triplicate samples and are representative of three separate experiments. The asterisk indicates significant differences ( p < 0.05) between the control and COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Real-time Polymerase Chain Reaction, Control, Clone Assay

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: The effect of COUP-TFI on the CXCL12/CXCR4 axis is mediated by EGF/EGFR activation. (A) The relative expression of EGF and EGFR mRNA was monitored by a real-time PCR analysis using MCF-7 control (Cont.) and COUP clones. The results were normalized against GAPDH as the internal control and are expressed as the mean EGF or EGFR mRNA/GAPDH mRNA ratio ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (B) ERK activation was examined in the MCF-7 control (Cont.) and COUP clones after a 5- or 10-min stimulation with EGF (10 −9 M) or CXCL12 (200 ng/mL). Western blots were performed using antibodies against phospho-ERK (P-ERK) and total ERK (ERK1/2); a representative western blot is presented. The importance of EGFR-specific signaling and general ERK signaling on CXCL12 (C) and CXCR4 (D) regulation was assayed by treating the cells with EGF (10 −9 M), AG1478 (25 μM), or U0126 (25 μM) for 48 h. The CXCL12 and CXCR4 relative mRNA levels were monitored by the real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Clone Assay, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI overexpression influences cellular responses to CXCL12. (A) The relative growth of the control (Cont.) and COUP clones was assayed with or without CXCL12 treatment for 7 days. The basal and CXCL12-induced cell growth were evaluated by MTT assays (n = 6) and determined in three independent experiments. The results are expressed as the relative cell number obtained when the control cells were treated with the vehicle control. Significant differences between the unstimulated control cells and the other conditions ( p < 0.05) are indicated with an asterisk. Significant differences between stimulated control cells and stimulated COUP clones ( p < 0.05 ) are indicated with a pound sign. (B) The migratory capacity of control (Cont.) and COUP clones was analyzed. The cells were maintained in phenol red-free DMEM/2.5% dsFBS for 48 h and then seeded in phenol red-free DMEM/0.5% dsFBS in the upper chamber of a PET 8-μm pore insert. The cells were allowed to migrate for 24 h toward the phenol red-free DMEM/2.5% dsFBS medium complemented or not with CXCL12 (200 ng/mL) and AMD3100 (50 μM). (C) CXCL12 was also added to the culture medium in the upper chamber prior to migration. The results are expressed as the mean ± SEM of the relative number of migratory cells compared to the basal migration of the control cells measured in three independent experiments. The asterisks indicate significant differences ( p < 0.05) from the basal migration of the control clones. The pound sign indicates significant differences ( p < 0.05) between two conditions linked by black lines.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Over Expression, Control, Clone Assay, Migration

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Box plots of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA expression in breast cancer and normal tissue. CXCR4 (A) , CXCR7 (B) , CXCL12 (C) , and COUP-TFI (D) mRNA expression was measured by real-time PCR in 23 normal breast tissue samples (NT), in 20 SBR grades 1 and 2, and in 19 SBR grades 3. The expression level was normalized by 18S RNA expression and analyzed with IQ5 software (Bio-Rad). The data are presented as whisker plots in which the horizontal bar represents the median, the grey boxes are the 25 th and 75 th percentiles, the vertical bar is the standard deviation, and the plus signs are the extreme points. All the Mann-Whitney tests were performed with Minitab 16 software, and the p value is indicated on the different graphs (ns denotes non-significant).

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Software, Whisker Assay, Standard Deviation, MANN-WHITNEY

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Sonic hedgehog is expressed in human brain arteriovenous malformations and induces arteriovenous malformations in vivo

doi: 10.1177/0271678x20912405

Figure Lengend Snippet: Figure 1. Immunohistochemical analysis of Shh pathway expression in human brain AVMs. Representative images of human brain AVMs and control brain vessels. Cells in the intimal layer of AVMs are strongly immunopositive for Shh and Gli1. COUP-TFII immunopositive cells are detectable in the intimal and medial layers of AVMs. Control brain vessels are negative in terms of Shh, Gli1, and COUP-TFII expression. Shh: Sonic hedgehog.

Article Snippet: For the detection of COUP-TFII expression in brain vasculature, sections were incubated with lectin from Lycopersicon esculentum (tomato) biotin conjugate (1:500) (SigmaAldrich, St. Louis, MO, USA) together with a rabbit polyclonal anti-COUP-TFII antibody (Origene, Rockville, MD, USA).

Techniques: Immunohistochemical staining, Expressing, Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Sonic hedgehog is expressed in human brain arteriovenous malformations and induces arteriovenous malformations in vivo

doi: 10.1177/0271678x20912405

Figure Lengend Snippet: Figure 7. Expression of COUP-TFII in phShh-induced brain neovessels. Sections of brain hemispheres injected with phShh and empty plasmid were stained for DAPI (blue staining) to identify cell nuclei, lectin (red staining) to identify blood vessels, and COUP- TFII (green staining) to identify cells expressing this vascular differentiation marker. (a) COUP-TFII-positive cells were found in the neovessels grown in the brain in response to phShh injection, both at the level of the intimal layer and the vascular wall. (b) COUP- TFII-positive cells were virtually absent in brain hemispheres injected with the empty plasmid.

Article Snippet: For the detection of COUP-TFII expression in brain vasculature, sections were incubated with lectin from Lycopersicon esculentum (tomato) biotin conjugate (1:500) (SigmaAldrich, St. Louis, MO, USA) together with a rabbit polyclonal anti-COUP-TFII antibody (Origene, Rockville, MD, USA).

Techniques: Expressing, Injection, Plasmid Preparation, Staining, Marker