primary mouse anti nr2f2  (R&D Systems)

Journal: The EMBO Journal

Article Title: A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

doi: 10.1038/emboj.2010.319

Figure Lengend Snippet: Temporal expression of NR2F2, OCT4 and miR-302 during hESC differentiation. RUES2 hESCs were differentiated in non-conditioned medium as a monolayer (nCM 2D; left panels) or embryoid body (EBs; right panels) for the indicated time points. CM: undifferentiated cells. (A) RT–PCR analysis of mRNA levels of the indicated genes. Lane −, negative control (no template). miR-302 HT indicates the host transcript of the miR-302/367 cluster. ATP5O is a ubiquitous gene used for normalization. (B) Northern blot analysis of mature miR-302a expression. The ubiquitous U2 snRNA is used for normalization. (C) Western blot analysis of protein levels. The anti-NR2F2 antibody recognizes an epitope in the N-terminal region of the full-length NR2F2 isoform. α-Tubulin is used for normalization. (D) Relative quantification by real-time PCR of mRNA and miRNA levels in A, B and by densitometric scanning of western blots in C (arbitrary units).

Article Snippet: For western blot, primary mouse anti-NR2F2 (H7147; R&D Systems), anti-Oct3/4 (BD611202; BD transduction lab) and anti-alpha/beta Tubulin (Cell Signaling) and secondary HRP-anti-mouse IgG (GE Healthcare) antibodies were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Northern Blot, Western Blot, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

doi: 10.1038/emboj.2010.319

Figure Lengend Snippet: NR2F2 is a bona fide miR-302 target. (A) HEK-293T cells were transfected with a vector encoding for full-length NR2F2 (coding sequence and untranslated regions) alone or in combination with either a synthetic miR-302a or a control non-targeting synthetic miRNA (miR-Co). First lane: mock-transfected cells. The figure shows western blot analysis of the indicated proteins. α-Tubulin is used for normalization. (B) Schematic representation of the reporter construct containing the Renilla luciferase-coding sequence fused to the NR2F2 3′ UTR. Numbers 1 and 2 refer to the predicted miR-302-binding sites. Sequences below illustrate the annealing of miR-302a to the wild-type NR2F2 (WT) and the point mutations (in red) introduced to generate the mutant NR2F2 3′ UTR (Mut1 and Mut2). (C) Luciferase assay. HEK-293T cells were transfected with the WT, single mutants (Mut1 and Mut2) or double mutant (Mut1+2) NR2F2 reporter genes in combination with synthetic miR-302a or control (miR-Co). Renilla luciferase activity was normalized with a co-transfected Firefly luciferase construct. Numbers above histogram bars indicate the fold change in luciferase activity. (D) Real-time PCR analysis of endogenous NR2F2 (left) or OCT4 (right) in RUES2 cells (blue bars) or miR-302 overexpressing RUES2-302 cells (red bars), differentiated as embryoid bodies for the indicated days. Asterisks in C, D indicate significant difference compared with the respective controls (Student's t-test; P<0.05).

Article Snippet: For western blot, primary mouse anti-NR2F2 (H7147; R&D Systems), anti-Oct3/4 (BD611202; BD transduction lab) and anti-alpha/beta Tubulin (Cell Signaling) and secondary HRP-anti-mouse IgG (GE Healthcare) antibodies were used.

Techniques: Transfection, Plasmid Preparation, Sequencing, Western Blot, Construct, Luciferase, Binding Assay, Mutagenesis, Activity Assay, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

doi: 10.1038/emboj.2010.319

Figure Lengend Snippet: OCT4 represses NR2F2 transcription in undifferentiated hESCs. (A, B) RT–PCR analysis of the indicated pluripotency (A) and differentiation (B) markers in untransfected (NT) RUES2 cells (green bars) or RUES2 cells transfected with non-targeting siRNAs (siCo, red bars) or anti-OCT4 siRNAs (siOCT4, blue bars). Note the log scale in B. Asterisks indicate significant difference compared with the respective NT controls (Student's t-test; P<0.05). (C) Western blot analysis of OCT4 and NR2F2 protein levels in the same conditions as in A and B. The anti-NR2F2 antibody recognizes an epitope in the N-terminal region of the full-length NR2F2 isoform. α-Tubulin is used for normalization. (D) Schematic representation of the 1.6 Kb OCT4-binding region located upstream of the NR2F2 locus (see also Figure 4A). Arrows indicate the position of primer sets 1 and 2 used in ChIP analysis. (E) Chromatin immunoprecipitation (ChIP) analysis in undifferentiated RUES2 cells. Chromatin was immunoprecipitated with the indicated antibodies (AcH3 is the acetylated form of histone H3; Rb IgG is a control normal rabbit IgG). DNA was then analysed for the enrichment of specific sequences, indicated on the left of each panel. POU5F1 is the locus encoding for OCT4.

Article Snippet: For western blot, primary mouse anti-NR2F2 (H7147; R&D Systems), anti-Oct3/4 (BD611202; BD transduction lab) and anti-alpha/beta Tubulin (Cell Signaling) and secondary HRP-anti-mouse IgG (GE Healthcare) antibodies were used.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation

Journal: The EMBO Journal

Article Title: A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

doi: 10.1038/emboj.2010.319

Figure Lengend Snippet: Alternative splicing generates several NR2F2 isoforms during hESC differentiation. (A) Schematic representation of the human NR2F2 locus (red box) on chromosome 15. Several isoforms generated by alternative splicing and alternative transcription start sites are depicted below. Black boxes indicate coding sequences (CDS), empty boxes indicate untranslated regions and lines represent introns. Arrows indicate transcriptional start sites. (B) RT–PCR analysis with isoform-specific primers in differentiated (nCM) and undifferentiated (CM) RUES2 cells. 2D, monolayer differentiation; EBs, embryoid bodies; NT, siCo and siOCT4 samples are the same as in Figure 3. ATP5O is used for normalization. (C, D) Luciferase reporter assay for transcriptional activation by NR2F2 isoforms in HeLa cells. In both panels, the Firefly luciferase activity is normalized with a co-transfected constitutive Renilla luciferase-encoding vector. (C) Cells were transfected with the NGFI-A:Luc reporter in combination with a control (GFP) or vectors encoding for the indicated NR2F2 isoforms. (D) Cells were co-transfected with the NGFI-A:Luc reporter and the full-length NR2F2-001 in combination with a control (GFP) or the NR2F2-203 or NR2F2-204-encoding vectors. In C and D, asterisks denote significant difference (Student's t-test; P<0.05) and numbers indicate fold change compared with the respective controls.

Article Snippet: For western blot, primary mouse anti-NR2F2 (H7147; R&D Systems), anti-Oct3/4 (BD611202; BD transduction lab) and anti-alpha/beta Tubulin (Cell Signaling) and secondary HRP-anti-mouse IgG (GE Healthcare) antibodies were used.

Techniques: Alternative Splicing, Generated, Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Assay, Activation Assay, Activity Assay, Transfection, Plasmid Preparation

Journal: The EMBO Journal

Article Title: A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

doi: 10.1038/emboj.2010.319

Figure Lengend Snippet: Spatial expression of NR2F2, OCT4 and Pax6 during hESC differentiation. (A–L′) Undifferentiated and (A–D) differentiated (7 days in non-conditioned medium as a monolayer; E–L′) RUES2 hESCs colonies were stained for the indicated proteins. (C, G, K, K′) Nuclear counterstain with Sytox Orange. I′–L′ are magnifications of white boxes in I–L, respectively. Scale bars represent 200 μm in A–L and 50 μm in I′–L′. (M) Real-time PCR analysis of the temporal expression of NR2F2 (blue line), Pax6 (red line) and Sox1 (green line) in RUES2 cells induced to differentiate into neural ectoderm. The protocol of differentiation is depicted below the histogram and described in detail in the Materials and methods section. Note the logarithmic scale. All measurements, except Sox1 D4, are statistically different compared with the undifferentiated (D0) sample (Student's t-test; P<0.01). (N–Q) RUES2 cells differentiated in N2M for 10 days with the protocol showed in M were stained for NR2F2 (N) and Pax6 (O), and counterstained with Sytox Orange (P). Scale bars represent 50 μm.

Article Snippet: For western blot, primary mouse anti-NR2F2 (H7147; R&D Systems), anti-Oct3/4 (BD611202; BD transduction lab) and anti-alpha/beta Tubulin (Cell Signaling) and secondary HRP-anti-mouse IgG (GE Healthcare) antibodies were used.

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

Journal: The EMBO Journal

Article Title: A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

doi: 10.1038/emboj.2010.319

Figure Lengend Snippet: Functional analysis of NR2F2 in hESCs. (A) Real-time PCR analysis of the indicated neural markers in differentiating RUES2 cells. Control mock-transfected cells (NT, blue bars) and cells transfected twice with non-targeting siRNAs (siCo, red bars) or anti-NR2F2 siRNAs (siNR2F2, green bars) were collected for analysis at day 7 of differentiation as a monolayer. (B) Differentiating RUES2 cells were transfected twice with control non-targeting antisense LNA oligonucleotides (LNA-C; blue bars) or with anti-miR-302 LNA (LNA-302; red bars). Cells were collected at day 8 and levels of indicated neural markers were assessed by real-time PCR analysis. Asterisks in A and B indicate significant difference compared with the respective controls (Student's t-test; P<0.05). (C) Schematic representation of the ePiggyBac-based inducible system for NR2F2 ectopic expression. TRE, TET responsive promoter; pA, polyadenylation signal. Pubc, human Ubiquitin C promoter; BsdR, Blasticidin resistance gene. In the ePB-TET-on vector, the constitutive CAG promoter drives a fusion gene encoding for the doxycycline-activated TET protein fused to the hygromycin resistance protein through a self-cleavage T2A peptide. (D–K) Immunostaining analysis of RUES2 cells stably transfected with the NR2F2-inducible system and left untreated (D–G) or cultured in presence of doxycycline (H–K). Cells were stained for OCT4 (D, H), NR2F2 (E, I) and counterstained with Sytox Orange (F, J). The scale bar in D represents 100 μm and can be applied to all panels.

Article Snippet: For western blot, primary mouse anti-NR2F2 (H7147; R&D Systems), anti-Oct3/4 (BD611202; BD transduction lab) and anti-alpha/beta Tubulin (Cell Signaling) and secondary HRP-anti-mouse IgG (GE Healthcare) antibodies were used.

Techniques: Functional Assay, Real-time Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation, Immunostaining, Stable Transfection, Cell Culture, Staining

Journal: The EMBO Journal

Article Title: A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

doi: 10.1038/emboj.2010.319

Figure Lengend Snippet: Model. The model illustrates different levels of regulation of pluripotency and neural differentiation mediated by the OCT4/NR2F2/miR-302 molecular circuitry in human embryonic stem cells. NR2F2 is repressed transcriptionally by OCT4 and post-transcriptionally by miR-302 and, in turn, inhibits OCT4 transcription. OCT4 activates transcription from the miR-302 locus and other pluripotency genes. NR2F2 is necessary for proper induction of neural genes during differentiation.

Article Snippet: For western blot, primary mouse anti-NR2F2 (H7147; R&D Systems), anti-Oct3/4 (BD611202; BD transduction lab) and anti-alpha/beta Tubulin (Cell Signaling) and secondary HRP-anti-mouse IgG (GE Healthcare) antibodies were used.

Techniques: