Demonstration of the interaction between ERRs and PNRC using mammalian two-hybrid assays. Demonstration of the interaction between ERRs and PNRC using mammalian two-hybrid assays. HeLa cells were transfected with reporter plasmid Gal4-LUC (0.5 μg) and PM-hERRα LBD (0.5 μg), PM-hERRβ LBD (0.5 μg), or PM-hERRγ LBD (0.5 μg), along with pVP-PNRC coactivator fragment (aa 270–327, 0.5 μg). Cells were incubated with 10 μm phytoestrogens or the same amount of DMSO for 24 h, washed twice with 1× PBS, and assayed for LUC activities. Each graph represents an average of three independent experiments. The results between the DMSO groups and the treatment groups were subjected to statistical analyses [Student's t test (unpaired)]. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Masatomo Suetsugi et al. Mol Cancer Res 2003;1:981-991 ©2003 by American Association for Cancer Research