Fig. 4. Osimertinib upregulates TNF expression in TP53-GOF mutant cells by inducing p53-p65 interaction.

a Schematic representation of ChIP-qPCR analysis. ChIP-qPCR primers were designed to amplify promoter (a) or control (b) regions of the TNF gene locus. Percentage of ChIP-qPCR amplicons derived from the promoter (b, d) or control (c, e) regions of TNF that were immunoprecipitated with antibodies to p65 (b, c) or to p53 (d, e), or with control IgG, from PC9/p53^WT, PC9/p53^V218del, or PC9/p53^R248Q cells incubated with or without 600 nM osimertinib for 24 h. Data are means ± SEM of triplicates from one experiment and are representative of two independent experiments. Fluorescence microscopic images of p53-p65 complexes (yellow) detected by an in situ PLA for PC9/p53^EV, PC9/p53^WT, and PC9/p53^R248Q cells under basal conditions (f) or for PC9/p53^R248Q cells incubated in the absence or presence of osimertinib (600 nM) or TNF-α (1 ng/ml) for 24 h (i). Nuclei were stained with DAPI (blue). The representative images were obtained by optical sectioning. Scale bars, 20 μm. Number of p53-p65 complexes per cell (g, j) and percentage colocalization of PLA signals with DAPI staining (h, k) determined from z-projection images constructed by z-stacking of optical sections for cells as in (f) and (i), respectively. Data are means ± SEM (n = 9 fields including a total of at least 50 cells). ***p < 0.001, ****p < 0.0001, NS (one-way ANOVA followed by Tukey’s test).