Journal: Human molecular genetics

Article Title: WNT/β-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production.

doi: 10.1093/hmg/ddt484

Figure Lengend Snippet: Figure 5. b-Catenin controls CYP11B2 transcription through stimulation of NGFIB nuclear receptors expression (A and B) b-catenin knockdown decreases NURR1 and NUR77 expression. NURR1 and NUR77 expression levels were analysed by RTqPCR on mRNAs and by western blot on proteins extracts (bottom panels) from H295R transfected for 5 days with control siRNA (siGFP) or a siRNA to b-catenin (sibcat) and treated for 6 h with 10 nM Angiotensin II. (C and D) Wnt/b-catenin activationinduces Nurr1 and Nur77expression. Nurr1 and Nur77expressionlevels were analysedby RTqPCRonmRNA from Y1cells that were treated withDMSO (controlgroup)or 500 nM of BIO for 3 or 6 h. (E andF) Nurr1 and Nur77 expression levels are increased in the adrenals of mice with constitutive b-catenin activation. Expression of Nurr1 and Nur77 was analysed by RTqPCR on mRNAs extracted from 10-month-old wild-type (n ¼ 6) and DCat (n ¼ 6) mice. (G and H) NURR1 expression is up-regulated in APA. NURR1 and NUR77 expression levels were analysed by RTqPCR on mRNAs from 35 APA and 17 NAPA. (I and J) b-Catenin stimulates transcription of Nurr1 and Nur77. Nurr1 and Nur77 expression levels were analysed by RTqPCR on mRNAs from Y1 cells stimulated by DMSO (control group) or 500 nM BIO alone or in combination with 100 nM actinomycin D for 6 h. (K and L) b-Catenin binding to NURR1 and NUR77 regulatory regions is inhibitedby PKF115-584 in H295Rcells.H295Rcells were treated withDMSO (controlgroup)or 1.0 mM PKF115-584 for 30 h.After treatment,cells were fixed and sheared chromatin was immunoprecipitated with 5 mg of a control immunoglobulin (Ig) or 5 mg of an antibody to b-catenin (b). PCRs were performed with primers(arrows)flankingtwoconservedputativeLEF/TCF-bindingsitesinNURR1regulatoryregions(K)andfourpreviouslydescribedAP1-bindingsitesinNUR77 promoter (L). Boxed lanes represent control immunoprecipitation experiments performed with b-catenin antibody and primers located in the coding sequences of NURR1 and NUR77. (M and N) b-Catenin binding to Nurr1 and Nur77 regulatory regions is increased in adrenals from DCat mice. Adrenals from six wild-type and six DCat mice were fixed and the sheared chromatin was immunoprecipitated with control (Ig) or b-catenin (b) antibodies. PCRs were performed with primers flanking two conserved putative Lef/Tcf-binding sites in Nurr1 regulatory regions (M) and four conserved Ap1-binding sites in Nur77 promoter (N). Boxed lanes represent control immunoprecipitation experiments performed with b-catenin antibody and primers located in the coding sequences of Nurr1 and Nur77. Positions in the four cartoons are relative to the transcription start site. (O) NURR1 and NUR77 binding to CYP11B2 regulatory regions are inhibited by

Article Snippet: After centrifugation, nuclei pellet was resuspended in NLB buffer (50 mM Tris–HCl, 10 mM EDTA, 1% SDS) and incubated on ice for 45 min. Chromatin was then sheared to an average length of 500 pb and submitted to immunoprecipitation with 5 mg of antibodies directed againstb-catenin (610153, BD Biosciences Pharmingen), NURR1 (sc-991, Santa Cruz), NUR77 (sc-990, Santa Cruz) or non-immune IgG controls (Millipore).

Techniques: Expressing, Knockdown, Western Blot, Transfection, Control, Activation Assay, Binding Assay, Immunoprecipitation

Journal: Human molecular genetics

Article Title: WNT/β-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production.

doi: 10.1093/hmg/ddt484

Figure Lengend Snippet: Figure 6. b-Catenin stimulates CYP21 and AT1R expression. (A) b-Catenin induces transcription of Cyp21. Cyp21 expression was analysed by RTqPCR on mRNAs from Y1 cells stimulated by DMSO (control group) or 500 nM BIO alone or in combination with 100 nM actinomycin D for 6 h. Bars represent the mean relative quan- tification of at least four individual experiments performed in triplicate+standard deviation. (B) b-Catenin and NURs binding to CYP21 promoter is inhibited by PKF115-584. H295R cells were treated as described in Figure 5 and sheared chromatin was immunoprecipitated with 5 mg of a control immunoglobulin (Ig), 5 mg of an antibody to b-catenin (b, top panel), 5 mg of an antibody to NURR1 (1, bottom panel) or 5 mg of an antibody to NUR77 (77, bottom panel). PCRs wereperformedwithprimersflankingoneputativeLEF/TCF-bindingsiteandoneNBRE.Boxedlanesrepresentcontrolimmunoprecipitationexperimentsperformed with b-catenin and NURR1 antibodies and primers located in the coding sequence of CYP21. (C) b-Catenin and Nurr1 binding to mouse Cyp21 promoter is increased inDCatadrenals.Adrenalsfromsixwild-typeandsixDCatmicewerefixedandtheshearedchromatinwasimmunoprecipitatedwith5 mgofacontrolimmunoglobulin (Ig),5 mgofanantibodytob-catenin(b),5 mgofanantibodytoNurr1(1)or5 mgofanantibodytoNur77(77).PCRswereperformedwithonesetofupstreamprimers flankingacombinationofputativeLef/Tcf,NbreandSf-1-bindingsites(toppanel)andonesetofdownstreamprimersflankingputativeLef/TcfandNbre-bindingsites (bottom panel). Boxed lanes represent control immunoprecipitation experiments performed with b-catenin and NURR1 antibodies and primers located in the coding sequence of Cyp21. (D) b-Catenin binding to AT1R promoter is inhibited by PKF115-584. H295R cells were treated as described above and sheared chromatin was immunoprecipitated with 5 mg of a control immunoglobulin (Ig) or 5 mg of an antibody to b-catenin (b). PCRs were performed with primers flanking one putative LEF/TCF-binding site. Boxed lane represents a control immunoprecipitation experiment performed with b-catenin antibody and primers located in the coding se- quence of AT1R. (E) b-Catenin binding to At1a and At1b promoters is increased in DCat adrenals. Adrenals from six wild-type and six DCat mice were fixed and the sheared chromatin was immunoprecipitated with 5 mg of a control immunoglobulin (Ig), or 5 mg of an antibody to b-catenin. PCRs were performed with one set of primers flanking a putative Lef1-binding site in At1a promoter (left panel) and one set of primers flanking one putative Ap1 site in At1b promoter (right panel). Positions in the three cartoons are relative to the transcription start site. Boxed lanes represent control immunoprecipitation experiments performed with b-catenin antibodies and primers located in the coding sequences of At1a and At1b. All ChIP experiments were repeated at least three times. ‘In’ refers to 10% of the amount of chromatin that was loaded in each immunoprecipitation and serves as a reference (10% input). Statistical analysis in (A) was performed by one-way ANOVA followed by Tukey’s post hoc test. ∗P , 0.05; NS, not significant.

Article Snippet: After centrifugation, nuclei pellet was resuspended in NLB buffer (50 mM Tris–HCl, 10 mM EDTA, 1% SDS) and incubated on ice for 45 min. Chromatin was then sheared to an average length of 500 pb and submitted to immunoprecipitation with 5 mg of antibodies directed againstb-catenin (610153, BD Biosciences Pharmingen), NURR1 (sc-991, Santa Cruz), NUR77 (sc-990, Santa Cruz) or non-immune IgG controls (Millipore).

Techniques: Expressing, Control, Standard Deviation, Binding Assay, Immunoprecipitation, Sequencing

Journal: Human molecular genetics

Article Title: WNT/β-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production.

doi: 10.1093/hmg/ddt484

Figure Lengend Snippet: Figure 7. Synthetic representation of the main findings of this study. In a healthy adrenal (top panel), WNT signalling is maintained at a basal level by the action of SFRP2(andpresumablyother actorsof WNT signalling). Aldosterone secretionisstimulatedbyAngiotensinIIthroughacascade involvingbindingof Angiotensin II to its receptor AT1R, which stimulates NURR1 and NUR77 (NURs)expression. These in turn controlthe expression of CYP21 and CYP11B2, two enzymes essential foraldosterone production.Whetherb-cateninplaysa rolein thecontrolofaldosteroneproductioninahealthyadrenalisunknown.In aldosterone-producingtumours (bottom panel), down-regulation of SFRP2 (or other regulators) results in deregulated WNT/b-catenin activation (70% of APA). b-Catenin is constitutively bound to thepromotersofAT1R,NURR1andNUR77.Thisresultsinincreasedexpressionofthesegenes,whichinturnstimulatesexpressionofCYP21andCYP11B2,leadingto increased production of aldosterone.

Article Snippet: After centrifugation, nuclei pellet was resuspended in NLB buffer (50 mM Tris–HCl, 10 mM EDTA, 1% SDS) and incubated on ice for 45 min. Chromatin was then sheared to an average length of 500 pb and submitted to immunoprecipitation with 5 mg of antibodies directed againstb-catenin (610153, BD Biosciences Pharmingen), NURR1 (sc-991, Santa Cruz), NUR77 (sc-990, Santa Cruz) or non-immune IgG controls (Millipore).

Techniques: Expressing, Activation Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

doi: 10.1186/s11658-017-0046-0

Figure Lengend Snippet: Expression profile of the members of the NR4A sub-family in BeWo cells treated with forskolin. BeWo cells were treated with forskolin (25 μM) for various time periods, followed by analysis of NR4A sub-family members at the mRNA and protein levels. a – Transcript profiles of Nor-1, Nurr-1 and Nur-77 in BeWo cells at 0, 1, 2, 6, 24 and 48 h of forskolin treatment. Relative expression was normalized with 18S rRNA. Data are expressed as means ± s.e.m. of three independent experiments performed in triplicate. b – Densitometeric plots of Nor-1, Nurr-1 and Nur-77 using GAPDH as a loading control. Data are represented as means ± s.e.m. of at least three experiments. Representative blots for the same are also shown. p ≤ 0.05 is considered statistically significant

Article Snippet: After protein transfer, the nitrocellulose membrane was blocked in 5% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20; pH -7.4) and further incubated at 4 °C overnight with an optimized dilution of 1:1000 of rabbit polyclonal antibodies against Nor-1, Nur-77 (Thermo Fisher Scientific) and GAPDH (Cell Signaling Technology Inc.), and mouse monoclonal antibody against Nurr-1 (Thermo Fisher Scientific) in TBST containing 2.5% BSA.

Techniques: Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

doi: 10.1186/s11658-017-0046-0

Figure Lengend Snippet: Effect of Nor-1, Nurr-1 and Nur-77 silencing on forskolin-mediated BeWo cell fusion and hCG secretion. BeWo cell knockdown for Nor-1, Nurr-1 or Nur-77 were made using siRNA as described in the section. The efficacy of silencing of Nor-1, Nurr-1 or Nur-77 transcripts was confirmed via qRT-PCR using specific primers and was studied after 0, 2 and 48 h of forskolin (25 μM) treatment in control siRNA-transfected and Nor-1-, Nurr-1- or Nur-77-silenced BeWo cells. a , b and c – Comparisons of the transcript levels of Nor-1, Nurr-1 and Nur-77, respectively, in the control siRNA-transfected and silenced cells. Each bar represents relative expression after normalization with 18S rRNA and is expressed as means ± s.e.m. of three independent experiments performed in triplicate. The effect of Nor-1, Nurr-1 or Nur-77 silencing on forskolin-mediated BeWo cell fusion was studied at 48 h using desmoplakin I + II staining and hCG secretion was studied with ELISA. d – The fold change in forskolin-mediated BeWo cell fusion in Nor-1-, Nurr-1- or Nur-77-silenced cells and control siRNA-transfected cells. Values are shown as the means ± s.e.m. of three independent experiments. Representative images of desmoplakin I + II staining (green) are appended alongside. The scale bar represents 20 μm. e – hCG secreted by control and Nor-1-, Nurr-1- or Nur-77-silenced cells in response to forskolin treatment at 48 h and represented as means ± s.e.m. of three independent experiments performed in duplicate. p ≤ 0.05 is considered statistically significant

Article Snippet: After protein transfer, the nitrocellulose membrane was blocked in 5% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20; pH -7.4) and further incubated at 4 °C overnight with an optimized dilution of 1:1000 of rabbit polyclonal antibodies against Nor-1, Nur-77 (Thermo Fisher Scientific) and GAPDH (Cell Signaling Technology Inc.), and mouse monoclonal antibody against Nurr-1 (Thermo Fisher Scientific) in TBST containing 2.5% BSA.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

doi: 10.1186/s11658-017-0046-0

Figure Lengend Snippet: Effect of Nur-77 knockdown on hCG- and GnRH-mediated cell fusion and hCG secretion. BeWo cells were treated with either hCG (5 IU/ml) or GnRH (10 ng) for various times. Subsequently, total RNA and cell lysates were isolated and processed for qRT-PCR and Western blotting, respectively, to analyze the transcript and protein levels of Nor-1, Nurr-1 and Nur-77. a and b – qRT-PCR analysis of Nor-1, Nurr-1 and Nur-77 in the form of bar graphs of BeWo cells treated with hCG and GnRH, respectively. Relative expression was normalized with 18S rRNA. Data are expressed as means ± s.e.m. of three independent experiments performed in triplicate. c and d – Protein expression profiles of Nor-1, Nurr-1 and Nur-77 in BeWo cells after treatment with hCG and GnRH, respectively, with GAPDH used as an internal control. Values are expressed as means ± s.e.m. of the band intensity of three independent experiments. Representative blots for the same experiment are appended with the graphs. BeWo cells transfected with either Nur-77 siRNA or control siRNA were treated with either hCG (5 IU/ml) or GnRH (10 ng/ml). The fold change in fusion was estimated after 48 h using desmoplakin I + II staining and hCG secretion was analyzed in GnRH treated cells via ELISA. e – The effect of hCG and GnRH treatment in the control and Nur-77-silenced BeWo cells on fusion at 48 h. The data are expressed as means ± s.e.m. of three independent experiments. Representative images of desmoplakin I + II staining in green and DAPI in blue are appended alongside. The scale bar is 20 μm. f – hCG secreted by control and Nur-77-silenced BeWo cells in response to GnRH treatment at 48 h. Data are represented as means ± s.e.m. of three independent experiments performed in duplicate. p ≤ 0.05 is considered statistically significant

Article Snippet: After protein transfer, the nitrocellulose membrane was blocked in 5% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20; pH -7.4) and further incubated at 4 °C overnight with an optimized dilution of 1:1000 of rabbit polyclonal antibodies against Nor-1, Nur-77 (Thermo Fisher Scientific) and GAPDH (Cell Signaling Technology Inc.), and mouse monoclonal antibody against Nurr-1 (Thermo Fisher Scientific) in TBST containing 2.5% BSA.

Techniques: Isolation, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

doi: 10.1186/s11658-017-0046-0

Figure Lengend Snippet: Transcript profiles of NR4A members in BeWo cells transfected with Nor-1, Nurr-1 or Nur-77 siRNA after forskolin treatment. BeWo cells were silenced for Nor-1, Nurr-1 or Nur-77 using the respective siRNA and transcript levels of all the three members were assessed using qRT-PCR after 0, 2 and 48 h of forskolin (25 μM) treatment. a , b and c – qRT-PCR analysis of the transcript levels of all three members in BeWo cells with Nor-1, Nurr-1 and Nur-77 knockdown, respectively. Each bar represents the relative expression after normalization with 18S rRNA, expressed as means ± s.e.m. of three independent experiments performed in triplicate. p ≤ 0.05 is considered statistically significant

Article Snippet: After protein transfer, the nitrocellulose membrane was blocked in 5% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20; pH -7.4) and further incubated at 4 °C overnight with an optimized dilution of 1:1000 of rabbit polyclonal antibodies against Nor-1, Nur-77 (Thermo Fisher Scientific) and GAPDH (Cell Signaling Technology Inc.), and mouse monoclonal antibody against Nurr-1 (Thermo Fisher Scientific) in TBST containing 2.5% BSA.

Techniques: Transfection, Quantitative RT-PCR, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

doi: 10.1186/s11658-017-0046-0

Figure Lengend Snippet: Effect of simultaneous silencing of Nor-1, Nurr-1 and Nur-77 on forskolin- or hCG-mediated BeWo cell differentiation. BeWo cells were knocked down for Nor-1, Nurr-1 and Nur-77 simultaneously using siRNA as described in section. To confirm target-specific silencing of all three nuclear orphan receptors, qRT-PCR was performed at 0, 2 and 48 h after forskolin treatment in control siRNA-transfected and silenced cells. The effect of NR4A silencing on BeWo cell fusion and/or hCG secretion was studied after 48 h of forskolin and hCG treatment. a – Comparison of the transcript levels of Nor-1, Nurr-1 and Nur-77 in the control siRNA-transfected and NR4A-silenced cells. Each bar represents relative expression after normalization with 18S rRNA, expressed as means ± s.e.m. of three independent experiments performed in triplicate. b – The fold change in forskolin-mediated BeWo cell fusion in control siRNA-transfected cells and NR4A-silenced cells. Values are shown as means ± s.e.m. of three independent experiments. c – hCG secreted by control and NR4A-silenced cells on treatment with forskolin for 48 h. Data are represented as means ± s.e.m. of three independent experiments performed in duplicate. p ≤ 0.05 is considered statistically significant

Article Snippet: After protein transfer, the nitrocellulose membrane was blocked in 5% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20; pH -7.4) and further incubated at 4 °C overnight with an optimized dilution of 1:1000 of rabbit polyclonal antibodies against Nor-1, Nur-77 (Thermo Fisher Scientific) and GAPDH (Cell Signaling Technology Inc.), and mouse monoclonal antibody against Nurr-1 (Thermo Fisher Scientific) in TBST containing 2.5% BSA.

Techniques: Cell Differentiation, Quantitative RT-PCR, Transfection, Expressing

Journal: PPAR Research

Article Title: Inhibitory Effects of a Novel PPAR- γ Agonist MEKT1 on Pomc Expression/ACTH Secretion in AtT20 Cells

doi: 10.1155/2018/5346272

Figure Lengend Snippet: Effects of MEKT1 (time dependently), rosiglitazone, and pioglitazone on Nurr1, Nur77, and Tpit protein expression. (a) AtT20 cells treated with MEKT1 (M) at 10 μ M for 24 hours, 6 hours, and 3 hours, rosiglitazone (R) at 10 μ M for 24 hours, and pioglitazone (P) at 10 μ M for 24 hours, or 0.1% DMSO as control (C) for 24 hours. Optical density (OD) of Nurr1/Nur77 was shown in figure (b), Nur77 in figure (c), and TBX19 (Tpit) in figure (d). OD of Nurr1/Nur77, Nur77, and TBX19 (Tpit) were normalized by OD of actin. Results are expressed as percentages of control (100%).

Article Snippet: For the detection of NURR1, Nur77, and TBX19 (Tpit) protein the membrane was blocked with 1% BSA for 30 minutes and probed with the primary antibody for Nur77/Nurr1 antibody (SC-990, Santa Cruz Biotechnology); anti TBX19 antibody (GTX77878, GeneTex); Nur77 (ab13851, Abcam) diluted at 1 : 1000 with 1% BSA, for overnight at 4°C, and was thereafter incubated with anti-rabbit IgG, horseradish peroxidase (HRP) linked whole antibody from donkey (NA934V, GE Healthcare Life Sciences, Pittsburgh, PA) (1 : 5000) for 1 hour at room temperature.

Techniques: Expressing, Control

Journal:

Article Title: Deficiency of the NR4A Orphan Nuclear Receptor NOR1 attenuates Neointima Formation Following Vascular Injury

doi: 10.1161/CIRCULATIONAHA.108.822056

Figure Lengend Snippet: (A-B) Endothelial denudation injuries were performed in littermate NOR1+/+ and NOR1-/- mice. (A) Tissues were harvested after 28 days and NOR1, Nur77, and Nurr1 expression was analyzed by immunohistochemistry (scale bar 10 μM). (B) mRNA was harvested from sham-operated and injured vessels at the indicated time points and analyzed for NOR1, Nur77, and Nurr1 expression using real-time RT-PCR. Data are normalized to TFIIB mRNA expression levels and presented as mean ± SEM fold increase relative to sham-operated vessels (* P < 0.05 vs. time point 0 of the same genotype, 2-way ANOVA). (C-D) SMC were isolated from NOR1+/+ and NOR1-/- mice, serum-deprived for 48h, and stimulated with 10% FBS. (C) mRNA was harvested at the indicated time points and analyzed for NOR1, Nur77, and Nurr1 mRNA expression using real-time RT-PCR. Data are normalized to TFIIB mRNA expression levels and presented as fold increase relative to quiescent cells (* P < 0.05 vs. quiescent, 2-way ANOVA). (D) NOR1, Nur77, and Nurr1 protein expression was analyzed by Western blotting at the indicated time point. Cohybridization for GAPDH was performed to assess equal loading. The autoradiograms are representative of three independently performed experiments.

Article Snippet: Western blotting was performed as described 9 using the following antibodies: Ser807/811 Phospho-Rb (9308, Cell Signaling), NOR1 (PP-H7833, R & D Systems), Nurr1 (AF2156, R & D Systems), Nur77 (LS-B114, Lifespan Biosciences), cyclin D1 (05-815, Millipore), cyclin D2 (M-20) and GAPDH (FL 335) (Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Isolation, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

doi: 10.1186/s11658-017-0046-0

Figure Lengend Snippet: Expression profile of the members of the NR4A sub-family in BeWo cells treated with forskolin. BeWo cells were treated with forskolin (25 μM) for various time periods, followed by analysis of NR4A sub-family members at the mRNA and protein levels. a – Transcript profiles of Nor-1, Nurr-1 and Nur-77 in BeWo cells at 0, 1, 2, 6, 24 and 48 h of forskolin treatment. Relative expression was normalized with 18S rRNA. Data are expressed as means ± s.e.m. of three independent experiments performed in triplicate. b – Densitometeric plots of Nor-1, Nurr-1 and Nur-77 using GAPDH as a loading control. Data are represented as means ± s.e.m. of at least three experiments. Representative blots for the same are also shown. p ≤ 0.05 is considered statistically significant

Article Snippet: After protein transfer, the nitrocellulose membrane was blocked in 5% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20; pH -7.4) and further incubated at 4 °C overnight with an optimized dilution of 1:1000 of rabbit polyclonal antibodies against Nor-1, Nur-77 (Thermo Fisher Scientific) and GAPDH (Cell Signaling Technology Inc.), and mouse monoclonal antibody against Nurr-1 (Thermo Fisher Scientific) in TBST containing 2.5% BSA.

Techniques: Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

doi: 10.1186/s11658-017-0046-0

Figure Lengend Snippet: Effect of Nur-77 knockdown on hCG- and GnRH-mediated cell fusion and hCG secretion. BeWo cells were treated with either hCG (5 IU/ml) or GnRH (10 ng) for various times. Subsequently, total RNA and cell lysates were isolated and processed for qRT-PCR and Western blotting, respectively, to analyze the transcript and protein levels of Nor-1, Nurr-1 and Nur-77. a and b – qRT-PCR analysis of Nor-1, Nurr-1 and Nur-77 in the form of bar graphs of BeWo cells treated with hCG and GnRH, respectively. Relative expression was normalized with 18S rRNA. Data are expressed as means ± s.e.m. of three independent experiments performed in triplicate. c and d – Protein expression profiles of Nor-1, Nurr-1 and Nur-77 in BeWo cells after treatment with hCG and GnRH, respectively, with GAPDH used as an internal control. Values are expressed as means ± s.e.m. of the band intensity of three independent experiments. Representative blots for the same experiment are appended with the graphs. BeWo cells transfected with either Nur-77 siRNA or control siRNA were treated with either hCG (5 IU/ml) or GnRH (10 ng/ml). The fold change in fusion was estimated after 48 h using desmoplakin I + II staining and hCG secretion was analyzed in GnRH treated cells via ELISA. e – The effect of hCG and GnRH treatment in the control and Nur-77-silenced BeWo cells on fusion at 48 h. The data are expressed as means ± s.e.m. of three independent experiments. Representative images of desmoplakin I + II staining in green and DAPI in blue are appended alongside. The scale bar is 20 μm. f – hCG secreted by control and Nur-77-silenced BeWo cells in response to GnRH treatment at 48 h. Data are represented as means ± s.e.m. of three independent experiments performed in duplicate. p ≤ 0.05 is considered statistically significant

Article Snippet: After protein transfer, the nitrocellulose membrane was blocked in 5% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20; pH -7.4) and further incubated at 4 °C overnight with an optimized dilution of 1:1000 of rabbit polyclonal antibodies against Nor-1, Nur-77 (Thermo Fisher Scientific) and GAPDH (Cell Signaling Technology Inc.), and mouse monoclonal antibody against Nurr-1 (Thermo Fisher Scientific) in TBST containing 2.5% BSA.

Techniques: Isolation, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Staining, Enzyme-linked Immunosorbent Assay