Progesterone and progestational compounds attenuate tumor necrosis factor alpha– induced interleukin-8 production via nuclear factor kappaB inactivation in endometriotic stromal cells  Sayako Horie, M.D., Tasuku Harada, M.D., Masahiro Mitsunari, M.D., Fuminori Taniguchi, M.D., Tomio Iwabe, M.D., Naoki Terakawa, M.D.  Fertility and Sterility  Volume 83, Issue 5, Pages 1530-1535 (May 2005) DOI: 10.1016/j.fertnstert.2004.11.042 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Effects of P and related compounds on the interleukin-8 (IL-8) messenger RNA levels in endometriotic stromal cells. In the presence of E2 (10−7 mol/L), the confluent endometriotic stromal cells were treated with P (10−6 mol/L), danazol (10−6 mol/L), or dienogest (10−7 mol/L) in the medium, supplemented with 0.1% charcoal-stripped FCS and incubated for 24 hours. In regard to P, the effect of antiprogestin was also examined by adding RU486 (10−6 mol/L) 1 hour before P treatment. Tumor necrosis factor α (TNFα; 0.1 ng/mL) was added and incubated for 6 hours. Total RNA was isolated from the cells and analyzed by Northern blot analysis. Total RNA (5 μg) was size-fractionated on 1% formaldehyde-agarose gels, transferred to Hybond-N+ membrane, and then hybridized to the IL-8 probe. The visualization of ethidium bromide-stained 28S ribosomal RNA subunits was used for normalization. Fertility and Sterility 2005 83, 1530-1535DOI: (10.1016/j.fertnstert.2004.11.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Electrophoretic mobility shift assay for nuclear NF-κB with nuclear extracts obtained from ESCs. In the presence of E2 (10−7 mol/L), the confluent ESCs were treated with P (10−6 mol/L), danazol (10−6 mol/L), or dienogest (10−7 mol/L) in medium supplemented with 0.1% charcoal-stripped FCS and incubated for 24 hours. In regard to P, the effect of antiprogestin was also examined by adding RU486 (10−6 mol/L) 1 hour before P treatment. Tumor necrosis factor α (TNFα; 0.1 ng/mL) was added and incubated for 15 minutes. Nuclear protein extract (5 μg) was incubated with radiolabeled NF-κB oligonucleotide. The resulting complexes were separated on a 5% nondenaturing polyacrylamide gel. To test for specificity of NF-κB binding, we carried out supershift analysis with antibody against the p50 and p65 subunit of NF-κB and competition experiments with a 100-fold excess of unlabeled oligonucleotide. Fertility and Sterility 2005 83, 1530-1535DOI: (10.1016/j.fertnstert.2004.11.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Effects of P and related compounds on interleukin-8 (IL-8) protein production in endometriotic stromal cells. In the presence of E2 (10−7 mol/L), the confluent ESCs were treated with P (10−6 mol/L), danazol (10−6 mol/L), or dienogest (−7 mol/L) in the medium supplemented with 0.1% charcoal-stripped FCS and incubated for 24 hours. The effects of antiprogestin were also examined by adding RU486 (10−6 mol/L) 1 hour before drug treatment. Next, tumor necrosis factor &alpha (TNFα; 0.1 ng/mL) was added and incubated for 24 hours. Concentrations of IL-8 in the supernatants were measured by ELISA. The minimum detectable level of IL-8 was 10 pg/mL. Bars represent SEs. *P<.05 vs. adding TNFα plus E2. Fertility and Sterility 2005 83, 1530-1535DOI: (10.1016/j.fertnstert.2004.11.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions