Figure 1. FX5 was a GR antagonist. (A) Chemical structure of FX5. (B) Mammalian one-hybrid assay was carried out to detect the antagonistic activity of FX5 against GR. In the assay, HEK293T cells were co-transfected with the plasmids of pCMX-Gal4-GR-LBD, pUAS-TK-luc and pRL-SV40, and then treated with Dex (10 nM) and FX5 (1, 5 and 10 μM) for 12 h, while luciferase activity was finally measured. (C) HEK293T cells were co-transfected with plasmids of pCI-nGFP-GR (C656G), pGL3-GRE-Luc and pRL-SV40, and then incubated with 10 nM Dex and different concentrations of FX5 (1, 5 and 10 μM) for 12 h. Finally, transactivation activity of GR was measured by luciferase reporter assay. (D) Mouse primary hepatocytes were treated with Dex (10 nM) and FX5 (5 and 10 μM) for 6 h, and GR protein levels in cytoplasm and nucleus were then measured by western blot assay. Quantification results of cytoplasmic GR protein level to GAPDH (E) and nuclear GR protein level to Histone H3 (F). (G) FX5 inhibited the Dex-stimulated GR-GFP nuclear translocation. (H) Ratio of GFP fluorescence intensity of nuclear and cytoplasm. (I) Mouse primary hepatocytes were treated with Dex (10 nM) and different concentrations of FX5 (1, 5 and 10 μM) for 6 h, and mRNA levels of GR were measured by quantitative RT-PCR analysis. Values were mean ± S.E.M (n=3/group) (*P<0.05, **P<0.01 and ***P<0.001).