Cytokine array analysis was evaluated using a mouse cytokine array C1000 (RayBiotech, Peachtree Corners, GA). B16F10 NF-κB-FLuc dendra2 cells (1 × 106) were seeded in a 100-mm cell culture plate. Four days later when cells were about 95% confluent, cell culture media was removed and replaced with 5 ml of serum-free media with or without 600 μM HOCl and incubated at 37°C in a humidified 5% CO2 atmosphere for 5 hours. Naïve CD8+ T cells were isolated from the spleens of adult C57BL/6 mice using a CD8a+ T cell isolation kit (Miltenyi Biotec, Auburn, CA) and labeled with CD3-phycoerythrin and CD8a-APC (BioLegend, San Diego, CA) to confirm the isolation of T cells by flow cytometry at the South Campus Flow Cytometry and Cell Sorting Core at UT MD Anderson Cancer Center. Isolated T cells were incubated in DMEM with or without 600 μM HOCl and incubated at 37°C in a humidified 5% CO2 atmosphere for 5 hours. After 5 hours, the media was collected in a 15-ml Falcon tube and centrifuged at 2000 rpm at 4°C for 10 min. The supernatant was removed and used for the cytokine array studies (RayBiotech, Peachtree Corners, GA). RNA was isolated from B16F10 and CD8+ T cells using an RNeasy Mini Kit (Qiagen, Germantown, MD). RNA-seq and analysis were performed by Admera Health conducted by Genohub (Genohub, Austin, TX). Differential expression analysis used DESeq; significantly differentially expressed genes comparing HOCl-treated and control B16F10 cells or HOCl-treated and control CD8+ T cells were defined as those with an adjusted P value of <0.05. Functional analysis of significantly differentially expressed genes was done with the GO classification system.

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