Journal: Journal of Biological Chemistry

Article Title: The NR4A Orphan Nuclear Receptor NOR1 Is Induced by Platelet-derived Growth Factor and Mediates Vascular Smooth Muscle Cell Proliferation

doi: 10.1074/jbc.m603436200

Figure Lengend Snippet: FIGURE 1. NOR1 protein is expressed in SMC of human coronary artery atherosclerotic lesions. Paraffin- embeddedserialsectionsofnormalhumancoronaryarteries(A)andarteriescontainingatheroscleroticlesions of different stages (B–E) were stained for smooth muscle -actin (upper panels) and NOR1 (lower panels). Objective magnifications are 20 (left panels) and 100 (right panels).

Article Snippet: Paraffin-embedded sections were incubated with the rabbit polyclonal NOR1 antibody (IMG-71915; Imgenex, Inc.) followed by biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories).

Techniques: Staining

Journal: Journal of Biological Chemistry

Article Title: The NR4A Orphan Nuclear Receptor NOR1 Is Induced by Platelet-derived Growth Factor and Mediates Vascular Smooth Muscle Cell Proliferation

doi: 10.1074/jbc.m603436200

Figure Lengend Snippet: FIGURE 2. PDGF induces NOR1 expression through ERK/MAPK-depen- dent signaling pathways. SMC were serum-deprived for 48 h and stimu- latedwithPDGF(25ng/ml).A,mRNAwasisolatedattheindicatedtimepoints and analyzed for NOR1 expression by Northern blotting. Cohybridization for the housekeeping gene 36B4 was performed as internal control. B, whole cell proteins were isolated at the indicated time points and analyzed for NOR1 protein expression by Western blotting. Cohybridization for GAPDH was per- formed to assess equal loading. C, quiescent SMC were pretreated with wort- mannin (WT, 200 mM), PD98059 (PD, 10 mM), SB203580 (SB, 15 mM), and SP600125 (SP, 25 mM) for 30 min prior to stimulation with PDGF (25 ng/ml). RNA was isolated 2 h after stimulation and analyzed for NOR1 mRNA expression by Northern blotting. The autoradiograms shown are repre- sentative of three independently performed experiments using different cell preparations. FIGURE 3. The proximal NOR1 promoter confers the transcriptional reg- ulation in response to PDGF. A, SMC were transiently transfected with a 4.0-kb NOR1 promoter construct, serum-deprived, and stimulated with vehi- cle (white bars) or PDGF (25 ng/ml, black bars) for 6 h. B, SMC were transfected with the indicated 5-deletion series of the 4.0-kb NOR1 promoter. Serum- deprived cells were stimulated with either vehicle (white bars) or PDGF (25 ng/ml, black bars) for 6 h. Following stimulation, cells were harvested, and luciferase activities were analyzed. Transfection efficiency was adjusted by normalizing firefly luciferase activities to Renilla luciferase activities gener- ated by cotransfection with 10 ng of pRL-CMV. Data are presented as mean S.E. from three independently performed experiments (*, p 0.05 versus vehicle). C, schematic illustration of the three cAMP-responsive elements located in the NOR1 promoter at 79, 64, and 53 from the transcription initiation site.

Article Snippet: Paraffin-embedded sections were incubated with the rabbit polyclonal NOR1 antibody (IMG-71915; Imgenex, Inc.) followed by biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories).

Techniques: Expressing, Protein-Protein interactions, Northern Blot, Control, Isolation, Western Blot, Transfection, Construct, Luciferase, Cotransfection

Journal: Journal of Biological Chemistry

Article Title: The NR4A Orphan Nuclear Receptor NOR1 Is Induced by Platelet-derived Growth Factor and Mediates Vascular Smooth Muscle Cell Proliferation

doi: 10.1074/jbc.m603436200

Figure Lengend Snippet: FIGURE 4. PDGF-induced NOR1 expression is mediated through CREB-dependent transactivation of the NOR1 promoter. A, SMC were transiently transfected with 1.7-kb NOR1 promoter constructs bearing muta- tions of the CRE sites at 79 (Cremt1), 64 (Cremt2), and 53 (Cremt3). B, SMC were cotransfected with the 1.7-kb NOR1 promoter construct and an empty control vector or an expression vector overexpressing a dom- inant-negative CREB mutant (ACREB). Following transfection, cells were serum-deprived and stimulated with vehicle (white bars) or PDGF (25 ng/ml, black bars) for 6 h. Luciferase activities were analyzed as described in the legend to Fig. 3. Data are presented as mean S.E. from three independently performed experiments (*, p 0.05 versus vehicle). C, SMC were transfected either with scrambled siRNA or CREB siRNA, serum-deprived, and stimulated with PDGF (25 ng/ml) for 6 h. Whole cell lysates were analyzed for NOR1 and total CREB protein expression by Western blotting. Cohybridization for GAPDH was performed to assess equal loading.

Article Snippet: Paraffin-embedded sections were incubated with the rabbit polyclonal NOR1 antibody (IMG-71915; Imgenex, Inc.) followed by biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories).

Techniques: Expressing, Transfection, Construct, Control, Plasmid Preparation, Mutagenesis, Luciferase, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The NR4A Orphan Nuclear Receptor NOR1 Is Induced by Platelet-derived Growth Factor and Mediates Vascular Smooth Muscle Cell Proliferation

doi: 10.1074/jbc.m603436200

Figure Lengend Snippet: FIGURE5.PDGFstimulatesSer-133phosphorylationofCREBandbinding to the NOR1 promoter through the ERK-MAPK signaling pathway. A, serum-deprived SMC were stimulated with PDGF (25 ng/ml) and analyzed for Ser-133-phosphorylated CREB by Western blotting. Cohybridization for total CREB was employed as control. B, serum-deprived SMC were preincubated with vehicle or PD98059 (10 mol/liter) for 30 min and stimulated with PDGF (25 ng/ml) for 15 min. Whole cell lysates were analyzed for Ser-133-phospho- rylated CREB and phosphorylated p44/42 MAPK. Cohybridization for total CREBandtotalp44/42MAPKwasemployedascontrol.C,quiescentSMCwere stimulated with PDGF (25 ng/ml) for the indicated time points. Following chromatin immunoprecipitation (IP) using a Ser-133-phospho-CREB anti- body (4 g) or control IgG, PCR analysis was performed using primer pairs that cover the CRE sites between 79 and 46 of the NOR1 promoter. Total extract (Input) was used as positive PCR control. D, serum-deprived SMC were preincubated with vehicle or PD98059 (10 mol/liter) for 30 min prior to stimulation with PDGF (25 ng/ml) for 30 min. Chromatin immunoprecipita- tion of Ser-133-phosphorylated CREB to the endogenous NOR1 promoter was performed as described in C. All autoradiograms shown are representa- tive of three independently performed experiments. DMSO indicates Me2SO.

Article Snippet: Paraffin-embedded sections were incubated with the rabbit polyclonal NOR1 antibody (IMG-71915; Imgenex, Inc.) followed by biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories).

Techniques: Western Blot, Control, Chromatin Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: The NR4A Orphan Nuclear Receptor NOR1 Is Induced by Platelet-derived Growth Factor and Mediates Vascular Smooth Muscle Cell Proliferation

doi: 10.1074/jbc.m603436200

Figure Lengend Snippet: FIGURE7.NOR1expressionisrequiredforSMCproliferation.Mouseaortic SMC were isolated from littermate wild type mice and NOR1-deficient mice. Equal numbers of cells (0.5 105 cells/plate) were plated on 60-mm plates. A, SMCweremaintainedinDMEMsupplementedwith10%FBS.After2,4,6,and 8 days, the cells were harvested, and cell proliferation was analyzed by cell counting using a hemocytometer. Cell proliferation was expressed as fold induction compared with day 0 and presented as mean S.E. (*, p 0.05 versus NOR1/). B, serum-deprived SMC were stimulated with PDGF (25 ng/ml) for the indicated time points, and cell proliferation was analyzed by cell counting. Cell proliferation was expressed as fold induction compared withday0andpresentedasmeanS.E.(*,p0.05versusNOR1/).C,NOR1 wild type or NOR1-deficient SMC were transfected with eukaryotic expression vectors overexpressing either GFP as control or NOR1. Following transfection, cells were maintained in DMEM supplemented with 20% FBS, and cell prolifera- tionwasanalyzedafter3days.Proliferationwasexpressedasfoldinductioncom- pared with day 0 and presented as mean S.E. (*, p 0.05 versus day 0).

Article Snippet: Paraffin-embedded sections were incubated with the rabbit polyclonal NOR1 antibody (IMG-71915; Imgenex, Inc.) followed by biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories).

Techniques: Isolation, Cell Counting, Transfection, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: The NR4A Orphan Nuclear Receptor NOR1 Is Induced by Platelet-derived Growth Factor and Mediates Vascular Smooth Muscle Cell Proliferation

doi: 10.1074/jbc.m603436200

Figure Lengend Snippet: FIGURE 8. NOR1 stimulates SMC proliferation by regulating cyclin D1 and D2 expression. A, serum-deprived NOR1 wild type and NOR1/ SMC were stimulated with PDGF (25 ng/ml) for 6 h, and whole cell lysates were analyzed for NOR1 protein expression by Western blotting. B, serum-deprived NOR1 wild type and NOR1/ SMC were stimulated with PDGF (25 ng/ml) for 24 h and analyzed for cyclin D1 and D2 expression by Western blotting. Cohybrid- ization for GAPDH was employed as control to assess for equal loading.

Article Snippet: Paraffin-embedded sections were incubated with the rabbit polyclonal NOR1 antibody (IMG-71915; Imgenex, Inc.) followed by biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

doi: 10.1371/journal.pone.0091462

Figure Lengend Snippet: (A, B) NR4A3 mRNA levels in response to (A) different doses of TG and (B) a fixed TG dose at a series of time points. (C, D) Chop mRNA levels in response to (C) different doses of TG and (D) a fixed TG dose at a series of time points. Relative mRNA levels of NR4A3 and Chop were determined with real-time quantitative PCR. (E, F) Spliced XBP1 (sXBP1) mRNA formation in response to (E) different doses of TG and (F) a fixed TG dose at different time points. Two forms of XBP1 (a UPR molecule) were detected with reverse transcription PCR. (G) NR4A3 protein profile in response to a fixed TG dose at a series of time points assayed with western blotting. (H) Semi-quantitative analyses of NR4A3 protein in response to a fixed TG dose at a series of time points. Data are shown as mean ± S.E. (n = 4). * p<0.05, ** p<0.01 vs. con (the dissolvent [DMSO] control group) or 0 h (baseline).

Article Snippet: Unless otherwise specified, all other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd. Anti-NR4A3 monoclonal antibody (PP-H7833-00) was purchased from R&D Systems; NOR-1 (sc-30154) rabbit polyclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and HA-tagged antibody (TA-04), beta-actin antibody (TA-09), and all secondary horseradish peroxidase-conjugated antibodies were from Zhongshan Goldenbridge Biotechnology Co (Beijing, China).

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

Journal: PLoS ONE

Article Title: Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

doi: 10.1371/journal.pone.0091462

Figure Lengend Snippet: (A, B) NR4A3 mRNA levels in response to (A) different doses of PA and (B) a fixed PA dose at a series of time points. (C, D) Chop mRNA levels in response to (C) different doses of PA and (D) a fixed PA dose at a series of time points. Relative mRNA levels of NR4A3 and Chop were determined with real-time quantitative PCR. (E, F) Spliced XBP1 (sXBP1) mRNA formation in response to (E) different doses of PA and (F) a fixed PA dose at different time points. Two forms of XBP1 (a UPR molecule) were detected with reverse transcription PCR. (G) NR4A3 protein profile in response to a fixed PA dose at a series of time points assayed with western blotting. (H) Semi-quantitative analyses of NR4A3 protein in response to a fixed PA dose at a series of time points. Data are shown as means ± S.E. (n = 4). * p<0.05, ** p<0.01 vs. con. Con is either (A, C, E) the vehicle (0.5% bovine serum albumin [BSA]) or (B, D, F– H) the average basal level in 0.5% BSA at a series of time points.

Article Snippet: Unless otherwise specified, all other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd. Anti-NR4A3 monoclonal antibody (PP-H7833-00) was purchased from R&D Systems; NOR-1 (sc-30154) rabbit polyclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and HA-tagged antibody (TA-04), beta-actin antibody (TA-09), and all secondary horseradish peroxidase-conjugated antibodies were from Zhongshan Goldenbridge Biotechnology Co (Beijing, China).

Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription, Western Blot

Journal: PLoS ONE

Article Title: Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

doi: 10.1371/journal.pone.0091462

Figure Lengend Snippet: (A, B) Relative mRNA levels of NR4A3 and Chop , respectively, determined with real-time quantitative PCR in response to different doses of tunicamycin (TM) in MIN6 cells. (D, E) Relative mRNA levels of NR4A3 and Chop , respectively, determined with real-time quantitative PCR in response to different doses of dithiothreitol (DTT) in MIN6 cells. (C, F) Spliced XBP1 (sXBP1) mRNA formation in response to different (C) TM or (D) DTT doses, respectively. Two forms of XBP1 (a UPR molecule) was detected with reverse transcription PCR. Data are shown as mean ± S.E. (n = 4). * p<0.05, ** p<0.01 vs. con (vehicle control group).

Article Snippet: Unless otherwise specified, all other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd. Anti-NR4A3 monoclonal antibody (PP-H7833-00) was purchased from R&D Systems; NOR-1 (sc-30154) rabbit polyclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and HA-tagged antibody (TA-04), beta-actin antibody (TA-09), and all secondary horseradish peroxidase-conjugated antibodies were from Zhongshan Goldenbridge Biotechnology Co (Beijing, China).

Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription, Control

Journal: PLoS ONE

Article Title: Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

doi: 10.1371/journal.pone.0091462

Figure Lengend Snippet: (A, B) Analysis of insulin secretion after glucose stimulation assayed by radioimmunoassay (RIA). (A) MIN6 cells were treated with 0.5 µM thapsigargin (TG) or DMSO (vehicle control) for 1 h, 0.5 mM palmitate (PA) or 0.5% BSA (vehicle control) for 12 h, and the supernatants were assayed for insulin protein level (n = 3). (B) MIN6 cells were infected with a series of double dilutions of Ad-NR4A3 adenovirus (adenovirus encoding NR4A3). Additional Ad-GFP (control adenovirus expressing GFP only) was used for complementary infection in order to ensure each infection had an equal virus titer. Post-infection, levels of secreted insulin were assayed by RIA (n = 4). Western blotting showed NR4A3 protein expression gradually decreasing, and insulin secretion level increasing accordingly. (C) mRNA levels of two insulin genes ( Ins1 and Ins2 ) were determined with reverse transcription PCR in MIN6 cells infected with Ad-NR4A3/Ad-GFP. (D) Semi-quantitative analyses of Ins1 and Ins2 mRNA levels in different adenovirus-infected MIN6 cells (normalized to beta actin) (n = 6). Data are shown as mean ± S.E. # p<0.05; **, ## p<0.01 vs. the control group or Ad-GFP infection only.

Article Snippet: Unless otherwise specified, all other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd. Anti-NR4A3 monoclonal antibody (PP-H7833-00) was purchased from R&D Systems; NOR-1 (sc-30154) rabbit polyclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and HA-tagged antibody (TA-04), beta-actin antibody (TA-09), and all secondary horseradish peroxidase-conjugated antibodies were from Zhongshan Goldenbridge Biotechnology Co (Beijing, China).

Techniques: RIA Assay, Control, Infection, Expressing, Virus, Western Blot, Reverse Transcription

Journal: PLoS ONE

Article Title: Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

doi: 10.1371/journal.pone.0091462

Figure Lengend Snippet: (A) Diagram of constructed wild-type and deletion forms of NR4A3 cDNA. (B) Verification by western blotting MIN6 cell lines stably over-expressing NR4A3 or its deletion forms. (C) mRNA levels of NR4A3 or two insulin genes ( Ins1 and Ins2 ) detected with reverse transcription PCR. Each image is representative of at least three experiments. (D) Semi-quantitative analyses of Ins1 and Ins2 mRNA levels normalized to beta actin in various stable cell lines (n = 5). ** P <0.01 vs. the control cells. Data are representative of three clone lines. Con, cell line transfected with vector encoding GFP; N, cell line expressing the wild-type NR4A3; ΔA, cell line expressing the 2–288 amino acid (aa) deletion of AF1 (activation function-1 domain); ΔD, cell line expressing the 292–364 aa deletion of the DNA binding domain (DBD); ΔL, cell line expressing the 398–626 aa deletion of the ligand binding domain (LBD). The C terminal of all exogenous genes was HA-tagged to facilitate identification with western blotting.

Article Snippet: Unless otherwise specified, all other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd. Anti-NR4A3 monoclonal antibody (PP-H7833-00) was purchased from R&D Systems; NOR-1 (sc-30154) rabbit polyclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and HA-tagged antibody (TA-04), beta-actin antibody (TA-09), and all secondary horseradish peroxidase-conjugated antibodies were from Zhongshan Goldenbridge Biotechnology Co (Beijing, China).

Techniques: Construct, Western Blot, Stable Transfection, Expressing, Reverse Transcription, Control, Transfection, Plasmid Preparation, Activation Assay, Binding Assay, Ligand Binding Assay

Journal: PLoS ONE

Article Title: Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

doi: 10.1371/journal.pone.0091462

Figure Lengend Snippet: Upon expression and activation of NR4A3 induced by factors such as long-chain free fatty acids (FFA) and thapsigargin (TG), which lead to ER stress, unfolded protein response (UPR) activation, and even apoptosis, this orphan nuclear receptor decreases insulin expression, which indirectly releases the burden of ER.

Article Snippet: Unless otherwise specified, all other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd. Anti-NR4A3 monoclonal antibody (PP-H7833-00) was purchased from R&D Systems; NOR-1 (sc-30154) rabbit polyclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and HA-tagged antibody (TA-04), beta-actin antibody (TA-09), and all secondary horseradish peroxidase-conjugated antibodies were from Zhongshan Goldenbridge Biotechnology Co (Beijing, China).

Techniques: Expressing, Activation Assay

Journal: Scientific Reports

Article Title: Extracellular vesicles derived from T regulatory cells suppress T cell proliferation and prolong allograft survival

doi: 10.1038/s41598-017-08617-3

Figure Lengend Snippet: dnIKK2-Treg-EV contain iNOS. ( A ) iNOS mRNA in dnIKK2-Treg-EV. Inducible NOS (iNOS) mRNA analysis in dnIKK2-Treg-EV and Trest-EV by real-time PCR. The cDNA content was calculated by ΔΔCt technique, using as calibrator the cDNA expression in Trest-EV. Results are expressed as arbitrary units (AUs), mean ± SD (n = 3 independent experiments). *p < 0.05 vs Trest-EV. ( B ) iNOS protein expression. iNOS protein expression in dnIKK2-Treg (left lane), or dnIKK2-Treg-EV (central lane), or LPS-stimulated rat peritoneal macrophages (MFs) as positive control (right lane), by Western blot. For each lane 20 μg of total proteins were loaded. Blot was cropped. Molecular weights are given on the left. One representative experiment of 3 is shown. ( C,D ) Expression and quantification of iNOS protein in allogeneic MLR performed in the presence of dnIKK2-Treg-EV. ( C ) iNOS protein expression (by Western blot) in protein extracts of cells from day 0 MLR (right lane) or day 3 MLR with (central lane) or without (left lane) dnIKK2-Treg-EV. For each lane 19 μg of total proteins were loaded. Blots were cropped. Molecular weights are given on the left. One representative experiment of 3 is shown. ( D ) results of densitometric analysis are given, after normalization with β-actin, as arbitrary unit (AU) considering naïve MLR as 1. Mean ± SD (n = 3 independent experiments).*p < 0.05 vs naïve Allo-MLR. ( E ) Effect of NOS inhibition on cell proliferation and IFN-γ + clone generation in dnIKK2-Treg-EV-exposed T cells. A 4-day Allo-MLR (1 × 10 6 LW lymph-node cells + 10,000 BN mature DC) was performed with or without EV from 20,000 dnIKK2-Treg (dnIKK2-Treg-EV) or Tact (Tact-EV) or Trest (Trest-EV) and + /− N-ω-nitro-L-arginine (NitroArg). Proliferation was measured by 3 H-Thymidine incorporation at day4 and expressed as cpm (left panel); frequency of IFNγ-producing T cells was assessed by ELISPOT (right panel). Results are expressed as mean ± SE (n = 3 independent experiments). *p < 0.05 vs all groups. # p < 0.05 vs ctr Allo-MLRs. ( F ) Effect of Carboxy-PTIO on cell proliferation in dnIKK2-Treg-EV-exposed T cells. A 4-day Allo-MLR was performed with or without EV from 20,000 dnIKK2-Treg (dnIKK2-Treg-EV) and + /− Carboxy-PTIO (5 μM). Proliferation was measured by 3 H-Thymidine incorporation at day4 and expressed as cpm.

Article Snippet: In selected experiments, MLR were performed in the presence of dnIKK2-Treg-EV and antibodies against Interleukin-12 (IL-12) (0.9 μg/ml, clone 20G101H7, Invitrogen) or IL-10 (1.45 μg/ml, clone 2G101H7, Invitrogen), or in the presence of N-ω-nitro-L-arginine (2 mM, Sigma-Aldrich,) – or of carboxy-PTIO (5 μM, Cayman Chemical).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Positive Control, Western Blot, Inhibition, Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands

doi: 10.1038/s41467-018-08069-x

Figure Lengend Snippet: Identification of recurrent rearrangements t(4;9)(q13;q31) in AciCCs. a Plots of copy number changes in four AciCCs with paired tumor-normal whole genome sequencing (WGS) show gains (green) and losses (red) in the individual tumors. b Circos plots of translocations in the four cases, with recurrent rearrangements t(4;9)(q13;q31) highlighted in red. c Upper panel: Detailed mapping of t(4;9)(q13;q31) chromosomal breakpoints of the four AciCCs with paired tumor-normal WGS and two AciCCs with only tumor WGS demonstrate the distribution of 4q13 breakpoints among ~ 340 kbps spanning eight different genes at the SCPP gene cluster (left side), and clustering of the 9q31 breakpoints upstream of the NR4A3 gene locus (right side). Middle panel: Detailed mapping of t(4;9)(q13;q31) chromosomal breakpoints of nine additional AciCCs with tumor hybrid capture sequencing data confirms the pattern of 4q13 breakpoints within the SCPP gene cluster (left) and upstream of the NR4A3 gene locus (right). Lower panel: SCPP gene cluster (left side), NR4A3 and neighboring genes (right side) with green and orange bars indicating the location of NR4A3 break apart FISH probes. d Absolute number of cases with genomic rearrangements of the NR4A3 gene locus in 28 AciCCs and 75 other salivary gland neoplasms analyzed by FISH. e mRNA expression (log2 FPKM values) of NR4A3 and neighboring genes in ten tumor samples and three normal parotid gland samples, with only NR4A3 showing a significant upregulation (deSeq2 ; *** P < 0.001; Box-plot center line: median; bounds of box: 25 and 75% quantiles; whiskers: minimum and maximum values). f NR4A3 immunohistochemistry demonstrating strong nuclear expression in AciCC tumor tissue (right), but absence in normal parotid gland tissue (left). Source data for Figs. b, and are provided as a Source Data file. Source Data for Fig. are provided in Supplementary Table . TCN, Tumor Copy Number; TXs, Translocations; SCPP, secretory Ca-binding phosphoprotein; AciCC, Acinar cell carcinoma; ACC, adenoid cystic carcinoma; BCA, basal cell adenocarcinoma; MASC, mamma-analog secretory carcinoma; MyoC, myoepithelial carcinoma; AdNOS, adenocarcinoma not otherwise specified; SDC, salivary duct carcinoma

Article Snippet: Immunohistochemistry was performed on 3 μm sections freshly cut from the TMA block using a fully automated staining system (“Benchmark XT System”, Ventana Medical Systems Inc, 1910 Innovation Park Drive, Tucson, Arizona, USA) and monoclonal antibodies against human NR4A3 (1:100 dilution, sc-393902 (H-7), Santa Cruz Biotechnology, INC., Heidelberg, Germany) against human Cyclin D1 (1:50 dilution, clone SP4, Zytomed, Berlin, Germany), and rabbit polyclonal against human Enolase 3 (Beta, Muscle) (1:2000 dilution, HPA000793, Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Techniques: Sequencing, Expressing, Immunohistochemistry, Binding Assay

Journal: Nature Communications

Article Title: Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands

doi: 10.1038/s41467-018-08069-x

Figure Lengend Snippet: Genomic breakpoints in AciCCs correlate with active chromatin marks and NR4A3 binding sites at the 4q13 SCPP gene cluster. Summary of (top to bottom) mRNA expression (log2(FPKM)), gene loci (Genes), genomic translocation breakpoints (TXs), copy-number variations (CNVs), active (H3K27ac, H3K4me3) and repressive (H3K27me3) histone marks, NR4A3 binding sites, CTCF binding sites, DNA methylation, topologically associated domains (TADs), and chromatin contacts (HiC) for the chromosomal regions surrounding the 4q13 (left panel) and 9q31 (right panel) breakpoints in normal parotid gland and AciCC tumor tissues. mRNA expression is shown for ten AciCC tumors and three normal parotid gland samples in red and blue, respectively (Box-plot centre line: median; bounds of box: 25 and 75% quantiles; whiskers: extend to last value greater than Q1–1.5*IQR, and last value less than Q3+1.5*IQR respectively. Here IQR is the inter quartile range, Q1 is the first, and Q3 the third quartile). Black bars demonstrating the translocations correspond to genomic material included in the rearrangement, with red and blue bars indicating gains and losses, respectively. For each ChIP-seq experiment, ChIP signals (barcharts), as well as corresponding peaks (directly below ChIP signals) are shown. For H3K27ac, the super-enhancer peaks are shown in addition (purple). Furthermore, CTCF motifs within CTCF peaks are shown (below CTCF peaks). Blue arrows indicate motifs on the forward strand, whereas red arrows indicate motifs on the reverse strand. The DNA methylation tracks show the average methylation within 1 kb binned regions. Different TADs are indicated in different colors

Article Snippet: Immunohistochemistry was performed on 3 μm sections freshly cut from the TMA block using a fully automated staining system (“Benchmark XT System”, Ventana Medical Systems Inc, 1910 Innovation Park Drive, Tucson, Arizona, USA) and monoclonal antibodies against human NR4A3 (1:100 dilution, sc-393902 (H-7), Santa Cruz Biotechnology, INC., Heidelberg, Germany) against human Cyclin D1 (1:50 dilution, clone SP4, Zytomed, Berlin, Germany), and rabbit polyclonal against human Enolase 3 (Beta, Muscle) (1:2000 dilution, HPA000793, Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Techniques: Binding Assay, Expressing, Translocation Assay, DNA Methylation Assay, ChIP-sequencing, Methylation

Journal: Nature Communications

Article Title: Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands

doi: 10.1038/s41467-018-08069-x

Figure Lengend Snippet: Detailed characterization of rearrangements t(4;9)(q13;q31) in 15 AciCCs identify three different breakpoint patterns. Detailed presentation of rearrangements in 15 AciCCs with t(4;9)(q13;q31), with black bars indicating genomic material included in the rearrangement. Active (H3K27ac, H3K4me3) and repressive (H3K27me3) histone marks, NR4A3 binding sites and CTCF binding sites in normal parotid gland tissue are presented for comparison. Source data for translocation breakpoints are provided as a file

Article Snippet: Immunohistochemistry was performed on 3 μm sections freshly cut from the TMA block using a fully automated staining system (“Benchmark XT System”, Ventana Medical Systems Inc, 1910 Innovation Park Drive, Tucson, Arizona, USA) and monoclonal antibodies against human NR4A3 (1:100 dilution, sc-393902 (H-7), Santa Cruz Biotechnology, INC., Heidelberg, Germany) against human Cyclin D1 (1:50 dilution, clone SP4, Zytomed, Berlin, Germany), and rabbit polyclonal against human Enolase 3 (Beta, Muscle) (1:2000 dilution, HPA000793, Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Techniques: Binding Assay, Comparison, Translocation Assay

Journal: Nature Communications

Article Title: Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands

doi: 10.1038/s41467-018-08069-x

Figure Lengend Snippet: Rearrangements t(4;9)(q13;q31) in AciCCs juxtapose the NR4A3 gene locus proximal to active enhancers and NR4A3 binding sites. Detailed presentation of active (H3K27ac, H3K4me3) and repressive (H3K27me3) histone marks, NR4A3 binding sites, CTCF binding sites, and DNA methylation levels in normal parotid gland ( a ) and three distinct AciCC tumor samples AciCC3 ( b ), AciCC1 ( c ) and AciCC2 ( d ). For each ChIP-seq experiment, ChIP signals (barcharts) as well as corresponding peaks (directly below ChIP signals) are shown. For H3K27ac, the super-enhancer peaks are shown in addition (purple). Furthermore, CTCF motifs within CTCF peaks are shown (below CTCF peaks). Blue arrows indicate motifs on the forward strand, whereas red arrows indicate motifs on the reverse strand. The DNA methylation tracks show the average methylation within 1 kb binned regions

Article Snippet: Immunohistochemistry was performed on 3 μm sections freshly cut from the TMA block using a fully automated staining system (“Benchmark XT System”, Ventana Medical Systems Inc, 1910 Innovation Park Drive, Tucson, Arizona, USA) and monoclonal antibodies against human NR4A3 (1:100 dilution, sc-393902 (H-7), Santa Cruz Biotechnology, INC., Heidelberg, Germany) against human Cyclin D1 (1:50 dilution, clone SP4, Zytomed, Berlin, Germany), and rabbit polyclonal against human Enolase 3 (Beta, Muscle) (1:2000 dilution, HPA000793, Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Techniques: Binding Assay, DNA Methylation Assay, ChIP-sequencing, Methylation

Journal: Nature Communications

Article Title: Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands

doi: 10.1038/s41467-018-08069-x

Figure Lengend Snippet: Functional impact of NR4A3 upregulation in AciCCs. a Hierarchical clustering of ten AciCCs and three normal parotid gland samples based on significantly up- and downregulated genes (deSeq2 ; FDR = 0.01). Each row represents a gene, and each column represents a sample, with color-coding of gene expression levels. With the exception of one case, the tumors cluster separately from the normal tissues. Functional enrichment analysis reveals seven annotation clusters among upregulated genes, and two annotation clusters among downregulated genes. Genes associated with annotation clusters and differential H3K27ac and NR4A3 peaks are indicated by bars. Red and blue bars encode up- and downregulated genes associated with functional annotation clusters (left), and genes associated with up- and downregulated H3K27ac and NR4A3 peaks (right), respectively. b The NBRE motif is the only significantly enriched known motif at sites with upregulated H3K27ac peaks in the tumor samples (HOMER ; P < 0.01). De-novo motif analysis of NR4A3 transcription factor ChIP-seq peaks in the tumor samples reveals the same NBRE motif in all three samples. c mRNA expression and d protein levels of NR4A3, Ccnd1, and Eno3 based on RNA sequencing and mass spectrometry analysis, respectively, in immortalized mouse submandibular gland cells transduced with NR4A3 or red firefly luciferase (RedFF) negative control. Box-plots and individual values for three replicates are shown (Box-plot center line: median; bounds of box: 25 and 75 % quantiles; whiskers: minimum and maximum values). e , f immortalized mouse submandibular gland cells and g , h human mammary MCF10A cells with and without overexpression of NR4A3 open reading frames were seeded in microscopy plates and incubated for the indicated times. e , g Cell numbers were counted using high-content screening microscopy after staining with Hoechst-33258. Individual values of six replicates for each time point are presented. f , h For cell cycle analysis, cells were labeled with BrdU and 7-AAD and analyzed by FACS. Averages and standard deviations as well as individual values of three replicates are shown. Two-tailed Student’s t -test; ** P < 0.01, *** P < 0.001. Source data for Figs. a and are provided as a Source Data file

Article Snippet: Immunohistochemistry was performed on 3 μm sections freshly cut from the TMA block using a fully automated staining system (“Benchmark XT System”, Ventana Medical Systems Inc, 1910 Innovation Park Drive, Tucson, Arizona, USA) and monoclonal antibodies against human NR4A3 (1:100 dilution, sc-393902 (H-7), Santa Cruz Biotechnology, INC., Heidelberg, Germany) against human Cyclin D1 (1:50 dilution, clone SP4, Zytomed, Berlin, Germany), and rabbit polyclonal against human Enolase 3 (Beta, Muscle) (1:2000 dilution, HPA000793, Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Techniques: Functional Assay, Gene Expression, ChIP-sequencing, Expressing, RNA Sequencing, Mass Spectrometry, Transduction, Luciferase, Negative Control, Over Expression, Microscopy, Incubation, High Content Screening, Staining, Cell Cycle Assay, Labeling, Two Tailed Test

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Signaling Cascade Mediating the Effect of FTY720P on the Na + /K + ATPase in LLC-PK1.

doi: 10.33594/000000561

Figure Lengend Snippet: Fig. 9. A) Effect of FTY720P (80 nM, 15 min) on the Na+/K+ ATPase activity in presence of carboxy-PTIO (30 nM). B) Effect of Glyco-SNAP-1 (4 µM, 15 min) on the Na+/K+ ATPase in presence of wortmannin (100 nM). Values are means ± SEM of 3 observations. Bars not sharing a common letter are considered significantly different from each other at P<0.001, as indicated by ANOVA followed by Tukey Kramer test.

Article Snippet: FTY720P, anti-p-Akt1/2/3 (Ser473)-R rabbit polyclonal antibody, Akt1/2/3 (H-136) rabbit polyclonal antibody, goat anti-mouse horseradish peroxidase (HRP) conjugated IgG, anti-GAPDH mouse monoclonal antibody, KT5823, carboxy-PTIO, glycol-SNAP-1,8-bromo-cGMP were purchased from Santa Cruz Biotechnology, CA, USA.

Techniques: Activity Assay