## 3 Results
### DHT Increases the Cytotoxicity of NK Cells  
In the above experiments, DHT increased NF-kB expression, increased the p65 enrichment to the _MICA_ promoter site, and ultimately increased the expression of MICA protein. MICA is a ligand for NKG2D, a NK-cell-activating receptor. The p65 ChIP-seq assay above showed that the p65 protein enriched at the promoter of RAET1G, another ligand of NKG2D, and _RAET1G_ transcription was also predicted to be increased (Supplementary Figure S2). Therefore, we hypothesized that the killing power of NK cells would be increased by upregulation of ligands of NK-cell receptors activating EBVaGC cells treated with DHT. To test this hypothesis, we investigated the cytotoxicity of NK-92-cells against DHT-treated SNU719 and YCCEL1 cells by lactate dehydrogenase (LDH) release-cytotoxicityassay. SNU719_AR(+) cells were treated with 100 nM DHT and control EtOH for 24 h, and then NK-92 cells were co-cultured with these SNU719_AR(+) cells for 24 h, and the cytotoxicity of NK-92 cells against SNU719_AR(+) cells was measured. NK-92 cytotoxicity was significantly higher in DHT-treated cocultures compared to the control, EtOH-treated cocultures (Figure 3A). Similarly, SNU719_AR(\(-\)) cells were treated with 100 nM DHT and control EtOH for 24 h, and then NK-92 cells were co-cultured with these SNU719_AR(\(-\)) cells for 24 h to measure the cytotoxicity of NK-92 cells against SNU719_AR(\(-\)) cells. NK-92 cytotoxicity was not statistically significant in DHT-treated co-cultures compared to the control, EtOH-treated co-cultures (Figure 3B). YCCEL1 cells were treated with 100 nM DHT and control EtOH for 24 h, and then NK-92 cells were co-cultured with these YCCEL1 cells for 24 h to measure NK-92 cell cytotoxicity against YCCEL1 cells. NK-92 cell cytotoxicity was significantly higher in DHT-treated cultures than in control EtOH-treated cultures (Figure 3C). Figure 3: **DHT increases the cytotoxicity of NK cells. ** (**A**) Investigation of cytotoxicity of NK-92 cells against SNU719_AR(+) cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicityicity of NK-92 cells against SNU719_AR(+) cells treated with EtOH for 24 h was used as a control. Statistically significant differences identified by the two-tailed Student’s _t_-test are indicated by asterisks (\({}^{\star}\)). (**B**) Investigation of cytotoxicity of NK-92 cells against SNU719_AR(\(-\)) cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicityicity of NK-92 cells against SNU719_AR(\(-\)) cells treated with EtOH for 24 h was used as a control. (**C**) Investigation of  
cytotoxicity of NK-92 cells against YCCEL1 cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of NK-92 cells against YCCEL1 cells treated with EtOH for 24 h was used as a control. (**D**) Analysis of transcriptional patterns of DHT-regulated NK-cell-activation receptor genes. The transcription of NK-cell-activation receptor genes in NK-92 cells treated with 100 nM DHT for 24 h was investigated by RT-qPCR assay. One-way ANOVA and [PERSON]'s multiple comparisons test were used for statistical processing. (**E**) Analysis of transcriptional patterns of NK-cell-activating receptor ligand genes regulated by DHT. The transcription of ligand genes of NK-cell-activating receptor in SNU719 cells treated with 100 nM DHT for 24 h was investigated by RT-qPCR assay. One-way ANOVA and [PERSON]'s multiple comparisons test were used for statistical processing. (**F**) Investigation of cytotoxicity of NK-92 cells against SNU719_PLKO.1 cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of NK-92 cells against SNU719_PLKO.1 cells treated with 100 nM DHT for 24 h was used as a control. (**G**) Investigation of cytotoxicity of NK-92 cells against SNU719_MICA(\(-\)) cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of NK-92 cells against SNU719_MICA(\(-\)) cells treated with EtOH for 24 h was used as a control.