pomc luc plasmid  (Addgene inc)

Journal: PLoS ONE

Article Title: Effects of RXR Agonists on Cell Proliferation/Apoptosis and ACTH Secretion/ Pomc Expression

doi: 10.1371/journal.pone.0141960

Figure Lengend Snippet: In A and B, total RNAs extracted from the cells cultured for 48 hr either in the presence (100 nM, 1 μM, 5 μM, or 10 μM) or absence (control) of each RXR agonist, HX630 and PA024, respectively, were examined for Pomc mRNA expression by quantitative real-time PCR. In C and D, supernatants obtained from the cells cultured for 48 hr either in the presence (100 nM, 1 μM, 5 μM, or 10 μM) or absence (control) of each RXR agonist, HX630 and PA024, respectively, were examined for ACTH secretion by EIA. In E and F, AtT20 cells transiently transfected for 24 hr with r Pomc -luc (-703 to +58-luc) and pCMV-β-gal were incubated either in the presence (100 nM, 1 μM, 5 μM, or 10 μM) or absence (control) of each RXR agonist, HX630 and PA024, respectively, before the luciferase assay. Results are expressed as percentages of each control. Each point represents mean ± SEM (n = 4). * P <0.05 vs control of each RXR agonist.

Article Snippet: Briefly, cells were transfected overnight with r Pomc -Luc reporter plasmid (-703 to +58-luc) using Lipofectamine LTX and Plus reagent (Invitrogen).

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Transfection, Incubation, Luciferase

Journal: PLoS ONE

Article Title: Effects of RXR Agonists on Cell Proliferation/Apoptosis and ACTH Secretion/ Pomc Expression

doi: 10.1371/journal.pone.0141960

Figure Lengend Snippet: A, effects of RXR agonists on the Pomc promoter deletion mutants in AtT20 cells. AtT20 cells transiently transfected for 24 hr with r Pomc -luc (-703 to +58-luc) or each deletion mutant reporter plasmid (-429 to +58-luc; -379 to +58-luc; -359 to +58-luc; -293 to +58-luc; -169 to +58-luc; -12 to +58-luc) and pCMV-β-gal were incubated either in the presence (10 μM) or absence (control) of each RXR agonist, HX630 and PA024, respectively, before the luciferase assay. Results are expressed as percentages of each control. Each point represents mean ± SEM (n = 4). * P <0.05 vs control in pGL3-Basic. B-G, effects of HX630 or PA024 on Nur77 , Nurr1 , and NeuroD1 mRNA expression in AtT20 cells. Total RNAs extracted from the cells cultured for 48 hr either in the presence (10 μM) or absence (control) of HX630 (B-D) or PA024 (E-G) were subjected to examine Nur77 , Nurr1 , and NeuroD1 mRNA expression by quantitative real-time PCR. Results are expressed as percentages of each control. Each point represents mean ± SEM (n = 4). * P <0.05 vs control of each RXR agonist. H, effects of HX630 on the binding of Nurr1/Nur77 heterodimer or NeuroD1 to the Pomc promoter. ChIP assays were performed using digested chromatin extracted from the cells cultured for 24 hr either in the absence (control) or presence (10 μM) of HX630 after transfection overnight with r Pomc -Luc reporter plasmid (-703 to +58-luc). Chromatin fragments were immunoprecipitated either by normal rabbit IgG (negative control), Nurr1/Nur77 antibody, or NeuroD1 antibody. Purified DNA was analyzed by conventional PCR using primers specific for both NurRE and E-box contained sequence in the rat Pomc promoter. The expected size for a fragment including NurRE and E-box is 211 bp (indicated in the lowest chart of Fig 4A). Few PCR products observed in the input samples were detected in the immunoprecipitation using normal rabbit IgG. I, effect of overexpression of Nurr1 or Nur77 on the HX630-mediated suppression of Pomc promoter activity. AtT20 cells transiently co-transfected for 24 hr with r Pomc -luc (-703 to +58-luc), pCMV-β-gal, Nurr1-pcDNA3, Nur77-pcDNA3, or pcDNA3 (Mock) were incubated either in the presence (10 μM) or absence (control) of HX630 for 24 hr, respectively, before luciferase assay. Results are expressed as percentages of the control. Each point represents mean ± SEM (n = 4). * P <0.05 vs Mock at 10 μM HX630.

Article Snippet: Briefly, cells were transfected overnight with r Pomc -Luc reporter plasmid (-703 to +58-luc) using Lipofectamine LTX and Plus reagent (Invitrogen).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Incubation, Luciferase, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Binding Assay, Immunoprecipitation, Negative Control, Purification, Sequencing, Over Expression, Activity Assay

Journal: PLoS ONE

Article Title: Effects of RXR Agonists on Cell Proliferation/Apoptosis and ACTH Secretion/ Pomc Expression

doi: 10.1371/journal.pone.0141960

Figure Lengend Snippet: A, effects of RXRα knockdown by its siRNA on the Pomc promoter activity. AtT20 cells transiently transfected with r Pomc -luc, pCMV-β-gal, and siRNA (negavive control or RXRα) for 48 hr were incubated either in the presence (10 μM) or absence (control) of HX630 for 24 hr, respectively. Results are expressed as percentages of each control. Each point represents mean ± SEM (n = 4). * P <0.05 vs negative control siRNA at 10 μM HX630. B, effect of RXRα overexpression on the Pomc promoter activity. AtT20 cells transiently transfected with r Pomc -Luc, pCMV-β-gal, and RXRα-pcDNA1/Amp (mRXRα) or pcDNA3 (Mock) for 24hr were incubated either in the presence (100 nM, 1 μM, 5 μM, or 10 μM) or absence (control) of HX630 for 24 hr respectively. Results are expressed as percentages of each control. Each point represents mean ± SEM (n = 4). * P <0.05 vs Mock control. ** P <0.05 vs RXRα control. C, effect of RXRα overexpression on Pomc mRNA expression. AtT20 cells transiently transfected with RXRα-pcDNA1/Amp (mRXRα) or pcDNA3 (Mock) for 24 hr were incubated either in the presence (10 μM) or absence (control) of HX630 for 24 hr, respectively. Results are expressed as percentages of each control. Each point represents mean ± SEM (n = 4). * P <0.05 vs RXRα control.

Article Snippet: Briefly, cells were transfected overnight with r Pomc -Luc reporter plasmid (-703 to +58-luc) using Lipofectamine LTX and Plus reagent (Invitrogen).

Techniques: Activity Assay, Transfection, Incubation, Negative Control, Over Expression, Expressing