## 3 Results
### Dht Regulates NF-\(\kappa\)B Activity
For the _AR_ control locus, which is a  
non-binding site for NF-\(\kappa\)B, IgG ChIP sample groupings are indicated by lowercase letters (a), and anti-p65 antibody ChIP sample groupings are indicated by lowercase letters (A\({}^{\prime}\)). (**F**) The p65 enrichment for upregulation of \(AR\) and \(RELA\). p65 ChIP-seq analysis was performed to evaluate the p65 enrichment sites in the genome of SNU719 cells treated with 10b0 nM DHT for 30 min. The reference human genome used was GRCH38/fg38. The p65 enrichment was assessed in the \(AR\) promoter locus located on chromosome X, q12 (**top**) and near the \(RELA\) promoter locus located on chromosome 11, q13.1 (**bottom**). 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. In the context of previous studies, we also wondered how DHT-AR signaling affects NF-\(\kappa\)B expression in EBVaGC cells, which we investigated as follows. First, we investigated how DHT regulates AR protein expression in SNU719 cells by Western blot assay. AR protein expression in SNU719 cells treated with DHT for 48 h showed a nearly concentration-dependent increase (Figure 1A). Third, we investigated whether DHT also enhances the activity of NF-\(\kappa\)B. SNU719 cells transfected with the pSI-Check2-hRluc-NF\(\kappa\)B-firefly reporter plasmid were treated with DHT for 6 or 24 h, and the activity of NF-\(\kappa\)B was measured by luciferase assay. The results showed that NF-\(\kappa\)B activity was statistically significantly increased in SNU719 cells treated with DHT for 6 h and 12 h, while NF-\(\kappa\)B activity was unchanged in SNU719 cells treated with DHT for 24 h (Figure 1C). Fourth, a previous study reported that AR could physically interact with NF-\(\kappa\)B in cells by co-immunoprecipitation (CO-IP) assay and that AR and NF-\(\kappa\)B can occupy the same DNA regulatory regions by ChIP assay [8]. This previous study led us to analyze whether DHT contributes to the interaction of AR with NF-\(\kappa\)B in EBVaGC cells by CO-IP and Western blot assays. We performed a Co-IP assay with anti-p52 Ab and Western blot assay with anti-AR Ab using SNU719 cells treated with 100 nM DHT for 48 h. We did not detect any interaction between AR and p52 proteins (Figure 1D). Moreover, we performed a Co-IP assay with anti-p65 Ab and Western blot assay with anti-AR Ab using SNU719 cells treated with 100 nM DHT for 48 h. We did not detect any interaction between AR and p65 proteins (Figure 1D). These results suggest that DHT increases the expression of AR and NF-\(\kappa\)B proteins but does not contribute to the interaction of AR with NF-\(\kappa\)B. Therefore, DHT-mediated AR signaling is indirectly related to the transcriptional activity of NF-\(\kappa\)B. Fifth, we wanted to further analyze whether AR and NF-\(\kappa\)B upregulated by DHT have a regulatory interaction with each other. [PERSON] et al. performed EMSA and showed that 1 h treatment with TNF-\(\kappa\)B binding to the \(MICA\) promoter, and that NF-\(\kappa\)B binding to the \(MICA\) promoter upregulated \(MICA\)[20]. Based on these experiments, we also investigated whether the p65 protein binds to the \(AR\) promoter locus after 1 h of DHT treatment by ChIP-qPCR assay (Figure 1E). The p65 protein was statistically significantly enriched at the \(AR\) promoter locus in SNU719_AR(+) cells treated with 100 nM DHT for 30 min. While there was also significant enrichment in the 60 min treatment group, the p65 protein was enriched much more strongly in the 30 min treatment group. These results suggest that DHT increases NF-\(\kappa\)B binding at promoter sites, which is essential as a transcriptional activator in SNU719 cells.