Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Menin and JunD regulate gastrin gene expression through proximal DNA elements.

doi: 10.1152/ajpgi.00160.2011

Figure Lengend Snippet: Fig. 1. Menin and JunD colocalize with gastrin in the stomach antrum. A: representative images of immunofluorescent staining in the wild-type mouse gastric antrum. Gastrin-producing G cells (green) expressed menin (red, left) and JunD (red, right). Nuclei were stained with DAPI (blue). Original mag- nification 600. B: AGS cells were transfected with menin expression vector for 48 h; 500 g of nuclear extracts were immunoprecipitated (IP) with either rabbit IgG or rabbit anti-JunD antibody. The immunoprecipitates were ana- lyzed by Western blotting using anti-menin and anti-GAPDH antibodies.

Article Snippet: 0193-1857/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpgi.org G783 at E B S C O on M ay 6, 2013 http://ajpgi.physiology.org/ D ow nloaded from Where indicated, the cells were treated after plasmid transfection with 10 nM of trichostatin A (Sigma-Aldrich, St. Louis, MO) for 28 h. AGS cells were plated (200,000 cells/ml) onto six-well plates in complete DMEM for 24 h, and then the media was replaced with serum-free Opti-MEM (Invitrogen) for 1 h. Duplex small-interfering RNAs (10 nM siRNA; Santa Cruz Biotechnology, Santa Cruz, CA) against menin (sc-35922) or JunD (sc-35728) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Staining, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Menin and JunD regulate gastrin gene expression through proximal DNA elements.

doi: 10.1152/ajpgi.00160.2011

Figure Lengend Snippet: Fig. 2. Reduced menin levels increase JunD protein levels. A: AGS cells were treated with 10 nM of scrambled (si-SCR, lanes 1 and 2) or menin (si-menin, lanes 3 and 4) siRNA. Whole cell extracts were analyzed by immunoblot. B: AGS cells were treated with increasing amounts of Octreotide (Oct) for 48 h, and whole cell extracts were analyzed by immunoblot analysis.

Article Snippet: 0193-1857/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpgi.org G783 at E B S C O on M ay 6, 2013 http://ajpgi.physiology.org/ D ow nloaded from Where indicated, the cells were treated after plasmid transfection with 10 nM of trichostatin A (Sigma-Aldrich, St. Louis, MO) for 28 h. AGS cells were plated (200,000 cells/ml) onto six-well plates in complete DMEM for 24 h, and then the media was replaced with serum-free Opti-MEM (Invitrogen) for 1 h. Duplex small-interfering RNAs (10 nM siRNA; Santa Cruz Biotechnology, Santa Cruz, CA) against menin (sc-35922) or JunD (sc-35728) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Western Blot

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Menin and JunD regulate gastrin gene expression through proximal DNA elements.

doi: 10.1152/ajpgi.00160.2011

Figure Lengend Snippet: Fig. 3. JunD and menin regulate gastrin gene expression in vitro. Shown are changes in gastrin mRNA expression determined by qRT-PCR. A: AGS cells were transfected for 48 h with the empty vector [pcDNA 3.1()], JunD, and menin expression vectors as indicated. *P 0.05. B: transient transfection of AGS cells for 72 h using scrambled (si-SCR) vs. menin (si-menin) or JunD (si- JunD) siRNA. *P 0.05. C: Western blot analysis of menin and JunD levels in the AGS cells after transfec- tion for 48 h with either vector (lane 1), JunD (lane 2), menin (lane 3), or JunD and menin together (lane 4). D: AGS cells were transfected for 72 h with either scrambled (lane 1), JunD (lane 2), or menin (lane 3) siRNA, and corresponding cell lysates were used for Western blot analysis. GAPDH is shown as a loading control.

Article Snippet: 0193-1857/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpgi.org G783 at E B S C O on M ay 6, 2013 http://ajpgi.physiology.org/ D ow nloaded from Where indicated, the cells were treated after plasmid transfection with 10 nM of trichostatin A (Sigma-Aldrich, St. Louis, MO) for 28 h. AGS cells were plated (200,000 cells/ml) onto six-well plates in complete DMEM for 24 h, and then the media was replaced with serum-free Opti-MEM (Invitrogen) for 1 h. Duplex small-interfering RNAs (10 nM siRNA; Santa Cruz Biotechnology, Santa Cruz, CA) against menin (sc-35922) or JunD (sc-35728) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Gene Expression, In Vitro, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Control

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Menin and JunD regulate gastrin gene expression through proximal DNA elements.

doi: 10.1152/ajpgi.00160.2011

Figure Lengend Snippet: Fig. 4. JunD activates the 9.8-, 3.2-, and 0.240-kb gastrin gene reporter constructs. A: 9.8-, 3.2-, and 0.240-kb GasLuc constructs or 0.240-kb GasLuc construct with mutations in the TGAC (TGAC), Sp1 (Sp1), gastrin epidermal growth factor response element (gERE) (gERE), or all three sites (combo) were cotransfected into the AGS cell line with pcDNA (set to 1), JunD alone, menin alone, or with JunD and menin expression vectors. *P 0.05. B: proximal promoter sequence of the human gastrin gene is shown. TGAC, Sp1, and gERE binding sites are underlined. C: chromatin immunoprecipita- tion assay analysis of the immunoprecipi- tated chromatin with either rabbit IgG, anti- JunD, anti-menin, or anti-Sp1 antibodies.

Article Snippet: 0193-1857/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpgi.org G783 at E B S C O on M ay 6, 2013 http://ajpgi.physiology.org/ D ow nloaded from Where indicated, the cells were treated after plasmid transfection with 10 nM of trichostatin A (Sigma-Aldrich, St. Louis, MO) for 28 h. AGS cells were plated (200,000 cells/ml) onto six-well plates in complete DMEM for 24 h, and then the media was replaced with serum-free Opti-MEM (Invitrogen) for 1 h. Duplex small-interfering RNAs (10 nM siRNA; Santa Cruz Biotechnology, Santa Cruz, CA) against menin (sc-35922) or JunD (sc-35728) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Construct, Expressing, Sequencing, Binding Assay

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Menin and JunD regulate gastrin gene expression through proximal DNA elements.

doi: 10.1152/ajpgi.00160.2011

Figure Lengend Snippet: Fig. 7. Trichostatin A (TSA) reversed the inhibitory effect of menin and JunD on the gastrin gene pro- moter. A: 0.240 GasLuc construct was cotransfected with either an empty vector, JunD alone, menin alone, or with JunD and menin expression vector and then treated with TSA. *P 0.05. B: shown are changes in gastrin mRNA expression determined by qRT-PCR. AGS cells were transfected with empty vector, with menin without and with JunD expres- sion vector for 48 h and then treated with TSA. *P 0.05. ns, not statistically significant. C: EMSAs with TGAC and Sp1 probe and nuclear extracts from AGS cells. The cells were transfected with empty vector, with menin without and with JunD expres- sion vector for 48 h and then treated with TSA.

Article Snippet: 0193-1857/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpgi.org G783 at E B S C O on M ay 6, 2013 http://ajpgi.physiology.org/ D ow nloaded from Where indicated, the cells were treated after plasmid transfection with 10 nM of trichostatin A (Sigma-Aldrich, St. Louis, MO) for 28 h. AGS cells were plated (200,000 cells/ml) onto six-well plates in complete DMEM for 24 h, and then the media was replaced with serum-free Opti-MEM (Invitrogen) for 1 h. Duplex small-interfering RNAs (10 nM siRNA; Santa Cruz Biotechnology, Santa Cruz, CA) against menin (sc-35922) or JunD (sc-35728) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Construct, Plasmid Preparation, Expressing, Quantitative RT-PCR, Transfection

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Menin and JunD regulate gastrin gene expression through proximal DNA elements.

doi: 10.1152/ajpgi.00160.2011

Figure Lengend Snippet: Fig. 8. Proposed model for menin and JunD regulation of the gastrin gene expression through proximal DNA elements. A: JunD binds the TGAC (non- canonical AP-1) element as well as Sp1 and gERE binding sites (via cooper- ation with Sp1) in the proximal gastrin gene promoter. JunD activates the gastrin gene promoter and induces basal levels of gastrin mRNA expression. B: Menin, possibly as part of the histone deacetylase (HDAC) complex, suppresses JunD-mediated gastrin gene activation. Rectangles (TGAC, Sp1 and gERE) show DNA-binding sites within the proximal gastrin promoter. Ovals indicate transcriptional factors.

Article Snippet: 0193-1857/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpgi.org G783 at E B S C O on M ay 6, 2013 http://ajpgi.physiology.org/ D ow nloaded from Where indicated, the cells were treated after plasmid transfection with 10 nM of trichostatin A (Sigma-Aldrich, St. Louis, MO) for 28 h. AGS cells were plated (200,000 cells/ml) onto six-well plates in complete DMEM for 24 h, and then the media was replaced with serum-free Opti-MEM (Invitrogen) for 1 h. Duplex small-interfering RNAs (10 nM siRNA; Santa Cruz Biotechnology, Santa Cruz, CA) against menin (sc-35922) or JunD (sc-35728) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Gene Expression, Binding Assay, Expressing, Histone Deacetylase Assay, Activation Assay