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. 2013 May 22;8(5):e64831. doi: 10.1371/journal.pone.0064831

Figure 6. Dimerization of GR is essential for ligand-independent loading of GR on endogenous GILZ gene promoter.

Figure 6

(A–B) COS-1 cells expressing GRwt (low, medium or high levels) or GRdim (low and medium levels) were induced for 8 hours with either ethanol or a range of DEX concentrations. RT-PCR of the GILZ gene was conducted and expression calculated relative to GAPDH for each condition. Sigmoidal dose-response curves were fitted to the experimental data. (A) Dose response curves of fold-induction of GILZ gene. (B) Summary table of GILZ transactivation results. Ligand-independent activation of GILZ is expressed relative to cells with no transfected GR. Statistical analysis of GILZ transactivation parameters comparing GRwt or GRdim to the low GRwt condition were carried out using one-way ANOVA followed by Dunnett's post-test (**P<0.01, ***P<0.001) and two tailed unpaired t tests of low GRwt against low GRdim or medium GRwt against medium GRdim (†P<0.05). (C–D) COS-1 cells transfected without or with GRwt (low, medium or high levels) or GRdim (low and medium levels) were induced for 1 hour with either ethanol or 10−6 M DEX. ChIP was performed using anti-GR antibody. RT-PCR of the promoter of the GILZ gene was performed on input chromatin, as well as the chromatin precipitated with anti-GR antibody. GILZ pull-down was normalized to input DNA. (C) GR recruitment to GILZ promoter expressed relative to the ethanol-induced no GR transfection condition. (D) DEX-induced fold-recruitment of GRwt to the GILZ promoter expressed relative to the ethanol condition within each transfection group. Statistical analysis of DNA-loading (C) comparing either ethanol- or DEX-induced conditions to their respective no GR transfected condition were carried out using one-way ANOVA followed by Dunnett's post-test (*P<0.05,**P<0.01, ***P<0.001). Fold-recruitment following DEX induction (D) statistical analysis comparing to low GRwt levels was through one-way ANOVA followed by Dunnett's post-test (*P<0.05). All results represent a minimum of three independent experiments performed in triplicate (±SEM).