Estrogen receptor β agonist diarylpropionitrile inhibits lipopolysaccharide-induced regulated on activation normal T cell expressed and secreted (RANTES) production in macrophages by repressing nuclear factor κB activation  Shi-ying Huang, M.S., Hong Xin, M.D., Jing Sun, M.D., Rui Li, M.S., Xue-mei Zhang, Ph.D., Dong Zhao, M.D.  Fertility and Sterility  Volume 100, Issue 1, Pages 234-240 (July 2013) DOI: 10.1016/j.fertnstert.2013.02.052 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 ERβ expression in RAW264.7 cells. (A) RAW264.7 and mouse ovary lysates were analyzed by RT-PCR for the presence of the mRNA encoding ERβ. (B) ERβ protein expression in mouse ovary, murine PMΦ, and RAW264.7 (RAW) lysates using Western blot analysis. Fertility and Sterility 2013 100, 234-240DOI: (10.1016/j.fertnstert.2013.02.052) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of DPN on RANTES production in murine RAW264.7 and RAW264.7 after ERβ siRNA transfection. (A) RANTES production was determined after a 2-hour pretreatment with saline, DPN, or dexamethasone (positive control), followed by treatment for 24 hours with LPS. (B) RANTES level after siERβ transfection. Nontargeting siRNA [RNAi(−)] was set as the negative control. RAW264.7 cells were pretreated with 10−7 mol/L DPN or saline, and after 24 hours LPS stimulation. The data reported are the mean ± SEM of three independent experiments, each performed in duplicate. *P<.05 as compared with the control group. (C) ERβ protein level in RAW264.7 after ERβ siRNA transfection. Fertility and Sterility 2013 100, 234-240DOI: (10.1016/j.fertnstert.2013.02.052) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 DPN suppresses p65 phosphorylation and IκB degradation in RAW264.7. (A) Effects of NF-κB signal on RANTES accumulation. Cells were pretreated for 2 hours with DPN (10−7 mol/L) or PDTC (10−5 mol/L) and then were stimulated with LPS for 24 hours. *P<.05 as compared with the control group. #P<.05 as compared with the LPS group. (B) Effect of DPN on LPS-induced NF-κB activation and p65 phosphorylation in time course. (C) Effect of DPN on IκB degradation in time course. (D, E) Quantitative data depicting the phospho-p65/total p65 ratio (D) and the IκB/β-actin ratio (E). Cells were pretreated for 2 hours with or without DPN (10−7 mol/L) and then were stimulated with LPS (1 μg/mL) for 0, 5, 10, 15, 30, and 60 minutes. The protein levels were quantitated by mean density value. The results are expressed as the mean ± SEM of four independent experiments, each performed in triplicate. *P<.05 compared with the LPS group at each respective time point. Fertility and Sterility 2013 100, 234-240DOI: (10.1016/j.fertnstert.2013.02.052) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 DPN suppressed NF-κB nuclear translocation in RAW264.7. (A) RAW264.7 cells were grown on glass coverslips and [1] left untreated, [2] treated with LPS for 15 minutes, or [3] pretreated with DPN for 2 hours, followed by 15 minutes of LPS stimulation. Cells were visualized using a fluorescent microscope (scale bar = 20 μm). (B) The relative intensity (nucleus/cytoplasm) of the p65 fluorescence in panel A was quantified. Over 80 cells were analyzed in each case. The results are expressed as the mean ± SEM of four independent experiments. *P<.05 compared with the control group. #P<.05 as compared with the LPS group. Fertility and Sterility 2013 100, 234-240DOI: (10.1016/j.fertnstert.2013.02.052) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions