Bz x analyzer software bz h4a

Two methods acquired images. One method was with a DM5500B fluorescent microscopy (Leica MICROSYSTEMS, Wetzlar, Germany) equipped with a HAMAMATSU ORCA-ER DIGITAL CAMERA (Hamamatsu Photonics, Hamamatsu, Japan) and processed with IPLab/Win (SOLUTION SYSTEMS, Funabashi, Japan). The other method was acquired with an all-in-one fluorescence microscope (BZ-8000, KEYENCE, Osaka, Japan) equipped with a Plan Apochromat 100× objective and processed with BZ-X Analyzer software (BZ-H4A, KEYENCE, Osaka, Japan).

PC9/p53^EV, PC9/p53^WT, and PC9/p53^R248Q cells were cultured to 70% confluence on 12-mm-diameter coverslips (Matsunami, Osaka, Japan) placed in 24-well plates (Corning, Corning, NY). PC9/p53^R248Q cells were treated with either DMSO as a control, 600 nM osimertinib, or TNF-α (1 ng/ml) for 24 h. Cells were fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 10 min with 0.3% Triton X-100 in PBS, and incubated overnight at 4 °C with 1:500 dilutions of mouse antibodies to p53 (#2524, Cell Signaling Technology) and rabbit antibodies to p65 (#8242, Cell Signaling Technology) for detection of p53-p65 complexes with the use of Duolink PLA Fluorescence Kits (#DUO92002 and #DUO92004; Sigma-Aldrich, St. Louis, MO, USA). Images of PLA signals were obtained with a BZX800 all-in-one fluorescence microscope (Keyence, Osaka, Japan). Optical sectioning images of p53-p65 complexes in multiple cells acquired from top to bottom of each cell were combined into a z-projection image with the use of full-focus imaging (BZ-H4A, Keyence). The number of PLA signals per cell and percentage of the signals in the nucleus stained with DAPI were quantified in nine fields including a total of at least 50 cells for each condition with the use of BZ-X Analyzer software (BZ-H4A, Keyence).

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).