FIGURE 2.

Effect of DIMN on androgen binding to AR and on androgen-induced AR activation steps. A, effect of DIMN on 5α-[³H]DHT binding to the AR. The binding inhibition was determined in COS-7 cells transiently transfected with pcDNA3.AR. The results are presented as percent binding relative to 5α-[³H]DHT alone and are shown for unlabeled DHT, BIC, OHF, and DIMN. B, inhibitory effects of DIMN on the AR N/C interaction. A mammalian two-hybrid assay was performed in PPC-1 cells transfected with 5XGAL4-Luc3, VP-AR1–660, and GAL-AR624–919. The interaction between the AR N and C termini was assessed after the addition of 10 μm of the indicated compound in the presence of 10 nm DHT. Error bars indicate the standard deviation. ***, p < 0.001. C, inhibitory effect of DIMN on the nuclear translocation of GFP-AR. HeLa cells transfected with the GFP-AR expression plasmid were grown on gelatin-coated coverslips. The subcellular localization of the GFP-AR in living cells was observed and recorded by fluorescence microscopy after a 1-h treatment with 10 μm BIC or DIMN in the presence of 10 nm DHT. D, inhibition of SRC-1- and SRC-2-mediated enhancement of AR transactivation by DIMN. 293T cells were co-transfected with pcDNA3.AR, pARE₂-TATA-Luc, and increasing amounts of pCR3.1 SRC-1 or pSG5-HA-SRC-2 (+, 300 ng; ++, 600 ng). After a 24-h transfection, cells were treated with 10 μm BIC or DIMN in the presence of 10 nm DHT. Each value represents the mean ± S.E. of at least three independent experiments. BIC and OHF were used as positive controls.