Tumor necrosis factor α up-regulates endometrial milk fat globule–epidermal growth factor 8 protein production via nuclear factor κB activation, resulting in cell migration of epithelial cells  Liang Yu, Ph.D., Sandra Anderson, B.S., Sergio Oehninger, M.D., Ph.D., Silvina Bocca, M.D., Ph.D.  Fertility and Sterility  Volume 101, Issue 2, Pages 552-559 (February 2014) DOI: 10.1016/j.fertnstert.2013.10.032 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Milk fat globule–epidermal growth factor 8 protein (MFG-E8) expression after tumor necrosis factor (TNF) α exposure. Ishikawa cells were treated with increasing doses of recombinant TNF-α for 24 hours. Both (A) ELISA and (B) Western blot analysis showed that MFG-E8 expression was up-regulated by TNF-α at ≥25 ng/mL compared with control. *P<.05. Fertility and Sterility 2014 101, 552-559DOI: (10.1016/j.fertnstert.2013.10.032) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 TNF-α regulates MFG-E8 via nuclear factor (NF) κB activation. Ishikawa cells were treated with NF-κB inhibitor Bay 11-7082 (Bay) at 5 μmol/L before addition of TNF-α at 50 ng/mL. (A) Quantitative analysis of p65 NF-κB localization after TNF-α treatment. Each bar indicates mean ± SEM of p65 nuclear positive cells out of the total cells (n > 300) counted from each experiment. ***P<.001 compared with control. (B) Representative images of NF-κB translocation in response to TNF-α treatment. Immunofluorescence staining was performed to identify NF-κB. Scale bar = 20 μm. (C) Western blot quantification showed MFG-E8 protein level was attenuated by pretreatment with Bay 11-7082. Each bar indicates mean ± SEM of protein expression relative to actin. *P<.05 compared with control. (D) Representative images of Western blot. Abbreviations as in Figure 1. Fertility and Sterility 2014 101, 552-559DOI: (10.1016/j.fertnstert.2013.10.032) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 MFG-E8 and inflammatory factors interleukin (IL) 6 and IL-8 mRNA expression was regulated by TNF-α and hCG. Ishikawa cells were treated with TNF-α (50 ng/mL) or with hCG (1,000 mIU) for 24 hours. Real-time polymerase chain reaction was used to analyze mRNA expression of (A) MFG-E8, (B) IL-6, and (C) IL-8. Each bar indicates fold increase of mRNA expression relative to housekeeping gene 18S. **P<.01 compared with control. Abbreviations as in Figure 1. Fertility and Sterility 2014 101, 552-559DOI: (10.1016/j.fertnstert.2013.10.032) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 TNF-α induces cell migration and MFG-E8 intracellular relocation. (A) Cell migration after 24 hours. Confluent monolayers of Ishikawa cells were wounded with a uniform scratch, washed to remove cell debris, and cultured for 24 hours in the absence or presence of 50 g/mL TNF-α or 2 μg/mL MFG-E8. Each bar indicates mean ± SEM of wound closure of Ishikawa cells treated without (Control) or with TNF-α or MFG-E8. **P<.011 compared with control. (B) Phase-contrast images of cell cultures were captured at 0 and 24 hours after scratching. Leading edges of the wounds are indicated by dotted lines. Scale bar = 1 mm. (C) E-Cadherin mediates TNF-α–induced migration in Ishikawa cells. Ishikawa cells were allowed to migrate for 24 hours after the treatment described above. Then the immunofluorescence images of E-cadherin were photographed. Arrows indicate areas in which the immunofluorescence signal is diminished. Scale bar = 20 μm. (D) TNF-α effects on MFG-E8 subcellular localization from cytoplasm extract (CE), membrane extract (ME), nuclear extract (NE), and pellet extract (PE) that contained solubilized cytoskeleton protein. Actin from equal percentages (by volume) of each fraction was used as loading control. Abbreviations as in Figure 1. Fertility and Sterility 2014 101, 552-559DOI: (10.1016/j.fertnstert.2013.10.032) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions