Bz h4a

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).

SMGs obtained from MyhCreER:tdTomato^fl/fl mice or SMG organ cultured tissues were fixed in 4% paraformaldehyde (Wako) at 4 °C for 1 h, followed by cryopreservation with sucrose (Wako). The cryosections (4 μm) were prepared and subjected to immunofluorescence using anti-mouse antibodies against FoxO1 (1:50; #2880; Cell Signaling Technology, Danvers, MA) and E-cad (1:100; #610181; BD Biosciences, Franklin Lakes, NJ), Eda (1:100; #PA5-72840; Invitrogen), Eda2r (1:100; #BS-7111R; Bioss), and phospho-NF-κB p65 (1:100; #3033; Cell signaling) overnight at 4 °C, followed by incubation with Alexa Fluor secondary antibody (1:200; Invitrogen) for 1 h at room temperature. Nuclei were counter-stained with DAPI (Dojindo, Kumamoto, Japan). Images were captured using a BZ-9000 fluorescence microscope (Keyence, Tokyo, Japan). FoxO1-positive areas were counted using a hybrid cell count application (BZ-H4C, Keyence) in BZ-X Analyzer software (BZ-H4A, Keyence).