AnAc 24:1ω5 inhibits E2-induced target gene transcription. To measure endogenous gene transcription, MCF-7, LCC9, LY2, MCF-10A, and MDA-MB-231 cells were serum-starved for 48 h and then treated for 6 h with EtOH, 10nM E2, and 10, 20, or 40 µM AnAc 24:1ω5 alone or in combination as indicated and as described in Materials and Methods. RNA levels of the target genes CCND1 (cyclin D1, A), CATD (cathepsin D, B) and TFF1 (pS2, C) were analyzed by real-time QRT-PCR as described in Materials and Methods. For MCF-10A and MDA-MB-231, CCND1 basal expression was ~25- and 114- fold higher than MCF-7 cells, respectively. For LY2, MCF-10A, and MDA-MB-231, CTSD basal expression was ~2-, 172-, and 138- fold higher than MCF-7 cells, respectively. No TFF1 expression was detected in cell lines other than MCF-7 (C). The effect of AnAc 24:1ω5 on each ER subtype (D) was examined in HEK-293 cells that were co-transfected with ERα (top) or ERβ (middle) in addition to an ERE-luciferase reporter and pRL-TK as described in Materials and Methods. MCF-7 cells (bottom) were transfected with the same ERE-luciferase reporter and pRL-TK as described in Materials and Methods. Twenty-four hours after transfection, the cells were treated with ethanol (EtOH), 10 nM E2 or the indicated concentrations of AnAc 24:1ω5 alone (solid lines, open squares) or in combination with 10 nM E2 (dashed lines, filled circles). Dual luciferase activity was assayed as described in Materials and Methods. Data are displayed as relative luciferase activity (fold difference) in which the EtOH activity was set to 1. For all panels, data are the mean ± SEM from 3 separate experiments. Values that were significantly different (P<0.05) from EtOH control are designated (a) and values from combined treatments that were significantly different (P<0.05) compared to E2 alone are designated (b).