## 3 Results
### DHT-Induced NF-\(\kappa\)B Uregulates MICA  
The p65 ChIP-seq assay described previously showed that the p65 protein binds to the promoters of various genes. As one of these, the p65 protein was found to be enriched at the MICA promoter locus (Figure 2A). The binding of the p65 protein to the \(MICA\) promoter locus was approximately three times weaker than the binding to its own \(RELA\) promoter locus. Figure 2: **DHT-induced NF-\(\kappa\)B regulates MICA. ****(A)** The p65 enrichment for the MICA upregulation. To investigate the p65 enrichment sites in the genome of SNU719 cells treated with 100 nM DHT for 30 min, we performed p65 ChIP-seq analysis. We examined the p65 binding pattern enriching the promoter of MICA located at p21.33 on chromosome 6. (**B**) Genomic structure of the MICA promoter locus (the 5\({}^{\prime}\) promoter site of MICA exon 1) expected to be bound by p65 protein and the MICA control locus (the 3\({}^{\prime}\) intron site of MICA exon 1) not expected to be bound by p65 protein. (**C**) Re-validation by ChIP-qPCR assay of p65 binding to the MICA promoter locus and the MICA control locus in SNU719_AR(+) cells. SNU719_AR(+) cells were treated with 100 nM DHT for 0, 30, and 60 min and then subjected to p65 ChIP-PCR assay. The p65 enrichment was examined at the MICA  
promoter locus, known to contain NF-kB binding sites, and the MICA control locus, known not to contain NF-kB nonbinding sites. ChIP assay results for the _MICA_ promoter locus, a known NF-kB binding site, are shown in the figure. Groupings of IgG ChIP samples are labeled with capitalized letters (A), while groupings of anti-p65 antibody ChIP samples are labeled with capitalized letters (A\({}^{\prime}\)). For the _MICA_ control locus, which is a non-binding site for NF-kB, IgG ChIP sample groupings are indicated by lowercase letters (A\({}^{\prime}\)), and anti-p65 antibody ChIP sample groupings are indicated by lowercase letters (A\({}^{\prime}\)). (D) Re-validation by ChIP-qPCR assay that p65 protein binds to the MICA promoter locus and the MICA control locus in SNU719_AR(\(-\)) cells. SNU719_AR(\(-\)) cells were treated with 100 nM DHT for 0, 30, and 60 min and then subjected to p65 ChIP-PCR assay. The p65 enrichment was examined at the MICA promoter locus and the MICA control locus. (E) Analysis of changes in the expression of 75-kDa and 65-kDa MICA/B proteins in SNU719 cells treated with different concentrations of DHT. (F) IFA assay detecting the increase in MICA expression induced by 1000 nM DHT treatment. We performed a one-way ANOVA test for comparisons involving more than three groups, followed by [PERSON]'s multiple comparison test if the results were significant. The results were presented using a compact letter display, with both uppercase and lowercase letters used to denote significant differences. As the binding of the p65 protein to the _MICA_ promoter locus was rather weak, we performed another p65 ChIP-qPCR assay under the same conditions. The p65 ChIP-qPCR assay examined the binding of the p65 protein to the _MICA_ promoter locus (5\({}^{\prime}\) promoter site of _MICA_ exon 1 (a 130 bp upstream region of _MICA_ exon 1 region) and the MICA control locus (3\({}^{\prime}\) intro site of _MICA_ exon 1, an intronic region adjacent to the 5\({}^{\prime}\) end of exon 2) (Figure 2B). In SNU719_AR(+) cells treated with 100 nM DHT for 30 min, the p65 protein bound statistically significantly to the _MICA_ promoter locus, and in SNU719_AR(+) cells treated for 60 min, the p65 protein bound statistically non-significantly to the _MICA_ promoter locus (Figure 2C). However, at the _MICA_ control locus, the p65 protein barely bound. Like SNU719_AR(+) cells, SNU719_AR(\(-\)) cells were treated with 100 nM DHT for 30 and 60 min, respectively, to investigate whether the p65 protein enriches at the _MICA_ promoter locus by p65 ChIP-qPCR assay (Figure 2D).