Scs n04

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).

An in situ apoptosis detection kit (Takara Bio, Shiga, Japan) was used for the analysis of LX-2 cells treated with HNK or vehicle according to the manufacturer’s instructions. Briefly, cells were incubated in chamber slides (SCS-N04, Matsunami Glass Ind., Osaka, Japan) with the indicated treatment for 24 h. The substrate 3,3′-diaminobenzidine (DAB) (Takara Bio, Shiga, Japan) was used for staining, which was observed with a BZ-X800 microscope. A total of 10 randomly selected fields of view at 200-fold magnification were used, and the number of the TUNEL-positive cells was averaged.