FIGURE 3.

Fra-1 is regulated by ESR1 during decidualization. Primary stromal cells isolated from day 4 pregnant mouse uterus were subjected to in vitro differentiation in the presence of P. A and B, cells were isolated to analyze the levels of Fra-1 (A, upper) and Esr1 (B, upper) mRNAs by real-time PCR. The protein levels of FRA-1 (A, lower) and ESR1 (B, lower) were analyzed by immunocytochemistry. C, endogenous expression of Esr1 mRNA was silenced by transfecting a siRNA specific to this transcript in mouse primary stromal cells. Cells were also transfected with a control scrambled (NC) siRNA. Mock represents treatment of cells with the transfection reagent without the oligonucleotides. After 48 h of transfection, RNA was subjected to real-time PCR, using gene-specific primers for Esr1, Pgr, Cx43, Fra-1, and GAPDH. The relative levels of gene expression in the Esr1 siRNA-treated samples compared with the mock or scrambled (NC) siRNA treatments are shown (left). Whole cell protein extracts obtained from Esr1 siRNA- and NC siRNA-treated cells were subjected to Western blot analysis using antibodies against FRA-1, ESR1, CX43, RNA polymerase II, and calnexin (right). D, endometrial stromal cells obtained from Esr1 siRNA- and NC siRNA-treated cells were subjected to immunocytochemical analysis using antibodies against ESR1 (upper) and FRA-1 (lower). Error bars, S.E.