## 3 Results  
### Dht Regulates NF-\(\kappa\)B Activity  
The androgen receptor (AR) and NF-\(\kappa\)B have complex interactions, which have been explored in various contexts in multiple studies. Key interactions include mutual inhibition, where AR activation can suppress NF-\(\kappa\)B transcriptional activity and vice versa [14; 15]. They also co-regulate certain target genes, often interacting in the regulation of genes related to inflammation, cell growth, and survival, with their effects varying depending on the cellular environment and specific stimuli [16]. In the tumor microenvironment, particularly in cancers such as prostate cancer, AR signaling can inhibit NF-\(\kappa\)B activity to exert anti-inflammatory effects, while NF-\(\kappa\)B activation can suppress AR signaling, promoting cell survival and growth [17; 18]. The interaction between AR and NF-\(\kappa\)B can differ across cell types and pathological conditions. Some studies show that in certain cancer cells, AR may regulate cell survival and inflammatory responses through NF-\(\kappa\)B activation [19]. These interactions are complex and dependent on various signaling pathways and intracellular environments, making them critical targets for disease treatment. Second, we evaluated the levels of NF-\(\kappa\)B p65 and its phosphorylated form (phospho-p65) in SNU719 cells following 48 h DHT treatment at varying concentrations. The expression of phospho-p65 and p65 proteins increased in the total lysate on treatment with 50-1000 nM DHT (Figure 1B). Similarly, we assessed the expression of phospho-p65, p65 and NF-kB p52 (p52) proteins in the nucleus of SNU719 cells treated with different concentrations of DHT for 48 h. The expression of phospho-p65, p65 and p52 proteins increased in the nucleus on treatment with 10-1000 nM DHT (Figure 1B). These results imply that DHT-mediated _AR_ overexpression is related to DHT-mediated NF-kB overexpression. Figure 1: **DHT regulates NF-kB activity. ** (**A**) _AR_ upregulation in SNU719 (**upper panel**) and MKN1-EBV (**bottom panel**) cells treated with DHT for 48 h. (**B**) Expression of the NF-kB p65 (p65) and NF-kB p52 (p52) proteins in the total lysate and the nucleus of SNU719 cells treated with 100 nM DHT for 48 h. Expression changes of phosphorylated p65 (p-p65) and p65 proteins in the total lysate were further investigated; p-p65 protein was p65 phosphorylated at serine 536 subunit. In addition, expression changes of p52 protein in the nucleus were additionally investigated. (**C**) Analysis of changes in NF-kB activity in DHT-treated SNU719 cells. The pSI-Check2-hRluc-NF-kB-firefly reporter plasmid was transfected into SNU719 cells for 48 h, then followed by 6 h (**left**), 12 h (**middle**) and 24 h (**right**) of DHT treatment, and finally luciferase activity was analyzed. (**D**) To analyze the interaction between NF-kB subunit (p52, p65) and AR proteins, a co-immunoprecipitation (co-IP)-Western blot assay was performed. First, IP was performed with anti-p52 Ab and Western blot assay was performed with anti-AR Ab (**left**). Next, IP was performed with anti-p65 Ab, and Western blot assay was performed with anti-AR Ab (**right**). (**E**) ChIP assay showing binding of the p65 protein to the _AR_ promoter locus (5\({}^{\prime}\) promoter site of _AR_ exon 1) and _AR_ control locus (3\({}^{\prime}\) intron site of _AR_ exon 1) in SNU719 cells. SNU719 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 _AR_ promoter locus, known to contain NF-kB binding sites, and the _AR_ control locus, known not to contain NF-kB nonbinding sites. ChIP assay results for the _AR_ 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\({}^{\prime}\)).