Figure 7

K18 modulates E2-activated reporter gene activity and the binding of LRP16 to ERα in MCF-7 cells. A, MCF-7 cells were grown in phenol-red free media stripped of steroids for at least 3 days, then cotransfected with 3×ERE-TATA-Luc reporter, ERα expression vector and the indicated vectors. 36 h after transfection, cells were treated with E2 (100 nM) or dimethyl sulphoxide (DMSO) for 6 h before luciferase assay. The relative luciferase activity levels were normalized by use of mock effector transfection and arbitrarily assigned a value of 1. All experiments were performed in triplicate and were repeated at least 3 times; results are expressed as means ± SEM. *, P < 0.05. B, MCF-7 cells were grown in phenol red-free media stripped of steroids for at least 3 days, then cotransfected with the indicated siRNA oligonucleotides, 3× ERE-TATA-Luc reporter and the ERα expression vector. 42 h after transfection, cells were treated with E2 (100 nM) or vehicle (DMSO) for 6 h before luciferase assay. Relative luciferase activity levels were normalized to transfections with control siRNA and were arbitrarily assigned a value of 1. All experiments were performed in triplicate and were repeated at least 3 times; results are expressed as means ± SEM. C, MCF-7 cells were transiently transfected with K18 expression vector or the corresponding empty vector. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (left panel). MCF-7 cells were cultured in phenol-red free media stripped of steroids for at least 3 days, then treated with E2 (100 nM) for 1 h and subjected to CoIP analysis by the use of the indicated antibodies (right panel). Ns-IgG, non-specific IgG.