Figure 5

Regulation of copper metabolism MURR1 domain (COMMD) and nuclear factor (NF)-ҡB by trophoblast-conditioned medium (TB-CM) in lipopolysaccharide (LPS)-induced human middle ear epithelial cells (HMEECs). (a) mRNA expression levels of the COMMD family (1–10) genes were examined by quantitative real-time polymerase chain reaction. (b) Significant upregulation was confirmed in the mRNA expression levels of COMMD1, COMMD3, and COMMD5. (c) Western blot analysis using an antibody of COMMD5 was used, which indicated the highest mRNA expression levels in the HMEEC nucleus and cytoplasm. There were no notable differences in the levels of COMMD5 protein expression in the cytoplasm. However, the protein expression levels of cell nuclei decreased and increased in the LPS and TB-CM groups, respectively. (d) mRNA expression levels of NF-ҡB decreased in a TB-CM concentration-dependent manner in LPS-induced HMEECs. (e) Proteins from the nucleus of cells were separated and subjected to Western blot analysis using NF-ҡB, pNF-ҡB, and β-actin antibodies. While the protein expression levels of pNF-ҡB increased in LPS-induced HMEECs, the protein expression level of pNF-ҡB significantly decreased when treated with TB-CM. β-actin expression levels were used to normalize all Western blot data. The outcomes of three separate trials are displayed as average ± standard deviation (SD). *P < 0.05, **P < 0.01, and ***P < 0.001.