Journal: Molecular Endocrinology
Article Title: Neurokinin B Causes Acute GnRH Secretion and Repression of GnRH Transcription in GT1-7 GnRH Neurons
Figure Lengend Snippet: NK3R may induce the long-term repression of GnRH transcription by activation of PKC and a closing of chromatin at the promoter of the GnRH gene. A, GT1-7 cells were transfected with NK3R and pretreated with DMSO or 10 μM of the broad spectrum PKC inhibitor Go6983 for 30 minutes before 8 hours cotreatment with vehicle, 30 nM senktide or 100 nM TPA. Luciferase values were internally normalized using a cotransfected β-galactosidase reporter to control for transfection efficiency, and data were normalized to an external control. Data are presented as mean fold ± SEM for n = 3 independent experiments. Comparisons were made using ANOVA with Least squares means Tukey HSD (α = .05). Levels not connected by same letter are significantly different. B, DNAse I Sensitivity Assay. GT1-7 cells were transfected with NK3R and treated with either vehicle or 30 nM senktide for 10 hours. DNA was isolated from nuclei and treated with 5 U of DNase I, then analyzed by quantitative PCR using primers specific to enhancer 1 or the promoter region of the GnRH gene (Supplemental Table 1). Amplicon quantities were normalized to undigested genomic DNA and are presented as the mean fraction of DNA remaining ± SD for n = 4 independent experiments. *Significantly different from vehicle-treated control by Student's t test ( P
Article Snippet: In brief, cell nuclei were resuspended in 1× DNase I Reaction Buffer (Promega) containing 2% glycerol, 5 U of DNase I (Promega) and incubated at 37°C for 5 minutes.
Techniques: Activation Assay, Transfection, Luciferase, Sensitive Assay, Isolation, Real-time Polymerase Chain Reaction, Amplification