Ca9 22

The human HNSCC cell lines, SAS and Ca9-22, were obtained from the RIKEN Bio-Resource Center (Tsukuba, Japan) and maintained in high-glucose Dulbecco’s modified eagle medium (DMEM; 6 Ltd., Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (P/S, Wako Pure Chemical Industries). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂.
Cells (1.0 × 10⁵) were seeded in 35 mm culture dishes (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and incubated for 6 h to allow for adherence to the surface. After incubation, the cells were exposed to X-rays. After 3 days or 6 days of continued culture at 37 °C, the cells were harvested using 0.25% trypsin-ethylenediaminetetraacetic acid (Wako Pure Chemical Industries, Ltd.) and the number of viable cells was counted using a trypan blue dye exclusion assay. For the 6-day cultures, the cells were collected 3 days after irradiation and re-seeded with cells (1.0 × 10⁵). After an additional 3 days of culturing, the cells were harvested for subsequent analysis.

A human gingival epithelial cell line (Ca9-22), obtained from RIKEN BRC (Tsukuba, Japan), was cultured in Dulbecco’s modified Eagle’s medium (Life Technologies Japan, Tokyo, Japan) supplemented with 10% fetal bovine serum (Life Technologies Japan), 50 U/mL penicillin and 50 µg/mL streptomycin solution (Life Technologies Japan) at 37 °C in 5% CO₂ atmospheric air. The cells from sub-confluent cultures were used after detachment with trypsin/EDTA (0.25%/1 mM). Titanium dioxide nano material (TTO-55 (A), nanotitania), with a size of <100 nm (mostly 30–50 nm), was obtained from Ishihara Sangyo Kaisha, Ltd. (Osaka, Japan). PgLPS was purchased from Wako (Osaka, Japan).