ERα-mediated transactivation of PLAC1 is dependent on NCOA3. (A) Quantitative real-time RT-PCR expression analysis of NCOA3 and PLAC1 in MCF-7 and SK-BR-3 cells 48 h after transfection with NCOA3- or PLAC1-specific siRNA duplexes. ns-siRNA, non-silencing siRNA. The results are shown as fold increase compared to untransfected cells used as a reference. (B) Protein expression of NCOA3 in MCF-7 and SK-BR3 cells 48 h after transfection with siRNA duplexes targeting NCOA3 or PLAC1 mRNA. Transfection of cells with PLAC1-specific siRNA was conducted to verify that knockdown of NCOA3 specifically affects PLAC1 mRNA and protein level. (C) Quantitative real-time RT-PCR analysis of PLAC1 expression in E2-depleted (72 h) MCF-7 cells in response to treatment with 100 nM E2, no E2 (C), or E2 and 5 μM ICI (E2/ICI), or ICI alone for 12 h. 48 h prior to treatment, cells were transfected with siRNA duplexes targeting NCOA3. Shown are mean and standard deviation of two independent experiments.