Bz h4c

A549-Dual cells were grown on four-well chamber slides (Matsunami Glass, SCS-N04) for 24 h before irradiation at a dose of 8 Gy. At 3 h post-irradiation, the cells were fixed with 4% PFA in phosphate-buffered saline (PBS) for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 3% BSA in PBS for 30 min at 37 °C. Following overnight incubation at 4 °C with the primary antibodies anti-phospho-H2A.X (Ser139) (CST, #2577) and anti-dsRNA-K1 (CST, 28764), the cells were incubated with a DyLight 488-conjugated donkey anti-mouse IgG secondary antibody (Invitrogen, SA5-10166) for 30 min at 37 °C. The primary and secondary antibodies were diluted 1:100 and 1:250, respectively, in PBS containing 3% BSA. The cells were then mounted with VECTASHIELD mounting medium with DAPI (H1200, Vector Laboratories). Images were obtained with an all-in-one fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan) equipped with a Plan Apochromat 40x objective (NA0.95, BZ-PA40, KEYENCE, Osaka, Japan). Positive areas and signal intensities were automatically calculated using a hybrid cell count application (BZ-H4C, KEYENCE, Osaka, Japan) in BZ-X Analyzer software (BZ-H4A, KEYENCE, Osaka, Japan).

HCEn cells were fixed in 4% paraformaldehyde and stained with a mouse anti-HLA-A antibody (BioLegend, San Diego, CA) and made visible with Alexa 555 conjugated secondary antibody (BioLegend). The cells were examined with an all-in-one fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) and analyzed using hybrid cell count software (BZ-H4C, Keyence, Osaka, Japan, https://www.keyence.com/ss/products/microscope/bz-casestudy/?tag_tips=Medical%20/%20Life%20Sciences).