FIGURE 4.

ICI-dependent LBD dimerization activity correlates with AF2ER activation. A, HepG2 cells were cotransfected with pG5-luc and expression vector for Gal4-DBD-fused WT-LBD (pBIND-LBD/WT) in the presence of expression vector for VP16-AD (pACT, left panel) or VP16-AD-fused WT-LBD (pACT-LBD/WT, right panel). The luciferase activity is represented as fold change over vehicle (0 nm) in the pACT and pBIND-LBD/WT co-transfected cells. B, HepG2 cells were cotransfected with pG5-luc and expression vector for Gal4-DBD-fused AF2ER-LBD (pBIND-LBD/AF2ER) in the presence of expression vector for VP16-AD (pACT, left panel) or VP16-AD-fused AF2ER-LBD (pACT-LBD/AF2ER, right panel). The luciferase activity is represented as fold change over vehicle (0 nm) in the pACT and pBIND-LBD/AF2ER co-transfected cells. Cells were treated with either vehicle (0 nm), E2 (0.01–10 nm, black column), or ICI (0.01–10 nm, white column). Luciferase activity is represented as mean ± S.D. a and c, p < 0.001 in each treatment against vehicle (0 nm) in the pACT and pBIND-LBD/WT co-transfected cells; b and d, p < 0.001 in each treatment against vehicle (0 nm) in the pACT-LBD/WT and pBIND-LBD/WT co-transfected cells; e, p < 0.001 in ICI treatment against vehicle (0 nm) in the pACT and pBIND-LBD/AF2ER co-transfected cells; e and f, p < 0.001 in ICI treatment against vehicle (0 nm) in the pACT-LBD/AF2ER and pBIND-LBD/AF2ER co-transfected cells. C, whole cell lysate was prepared from vehicle (V), E2 (1 nm), and ICI-treated (10 nm) transfected HepG2 cells and analyzed by immunoblotting with anti-ERα antibody (MC-20) to indicate the expression levels of Gal4-DBD/LBD and VP16-AD/LBD. β-actin (Actin) was used as a loading control. A representative Western blot analysis is shown.