Fig 2: Synergistic effects of gefitinib and JNJ on OVCAR3 cells with different p53 statuses were shown in Fa-CI plots and isobolograms. Based on the Chou and Talalay methods, the combination index (CI) values were calculated using CompuSyn software to determine the degree of synergistic effectiveness of the drug combination. The analysis revealed that overexpression of p53R248Q reduced the synergistic effect of gefitinib and JNJ. (A,B) In the Fa-CI plots, the fraction affected (Fa; from zero to one) represented the ratio of dead cells-to-total cells in response to drug treatments. Circled symbols indicated the CI values of each Fa. The CI values provided a quantitative definition of synergistic (CI < 1), additive (CI = 1), and antagonistic (CI > 1) effects derived from the drug combination. (G + J: gefitinib + JNJ) (C,D) In the isobolograms, the individual doses of JNJ (represented by x-axis) and gefitinib (represented by y-axis) that achieved 90% (Fa = 0.9), 75% (Fa = 0.75), and 50% (Fa = 0.5) inhibitory effects were indicated by green lines, red lines, and blue lines, respectively. Symbols (ED90 was marked as green triangles; ED75 was marked as red squares; ED50 was marked as blue circles) above the lines, on the lines, and below the lines represented antagonistic, additive, and synergistic effects, respectively. When the symbols were located below and distant from the lines, it represented strong synergism.

Fig 3: p53/caspase-8 death signaling involvement in Bad knockdown embryos at 24 hpf. (A) A network signaling death pathway was proposed based on accumulated data. The p53/caspase-8/tBid-mediated death pathway was induced. It also required upstream signaling from loss of Bad, leading to enhanced environmental stress with increased ROS and enhanced apoptotic cell death via the stress/death gene p53 and its correlated downstream gene expression caspase-8 or activation of tBid by cleavage from Bid. (B) Clear upregulation of p53 and caspase-8 demonstrated via qRT-PCR. All data were analyzed using either paired or unpaired Student’s t-tests, as appropriate. * p < 0.01. (C) Identification of apoptotic-related protein expression of p53/caspase-8/tBid as key molecules by Western blotting analysis at 24 hpf. Both p53 and caspase-8 showed stronger expression compared to the control group, correlated with downstream Bid cleavage and anti-apoptotic number Bcl-2 and Bcl-xL expression levels. (D) qRT-PCR evaluation of apoptosis-related gene expression with Bad knockdown in p53 mutant lines at 24 hpf. (E) Western blot analysis of apoptosis-related protein expression with Bad knockdown in p53 mutant fish lines correlated with downstream protein Bid cleavage and anti-apoptotic number Bcl-2 and Bcl-xL expression levels.

Fig 4: Brain development was directly restored during blockage of the p53-mediated stress signaling transition in p53 mutant lines with Bad knockdown. (A) Brain developmental marker pax2a expression in an in situ hybridization assay showing normal expression in the p53 mutant line (panels c and d) compared to the wild-type group (panels Aa–Ae) during Bad knockdown at 24 hpf. Defective marker expression was counted and is shown in panel (Ae). Scale bar = 100 µm. (B,C) The canopy-fused EGFP line was used to monitor brain development directly in the p53 mutant line during loss-of-Bad expression at 48 hpf. The midbrain/hindbrain boundary is indicated by an arrow. (C) Blockage of the p53-mediated stress signaling transition can restore brain development (indicated by arrows) compared to the wild-type (panels (Ca,Ce,Ci) for the Bad-M5 group; panels: (Cb,Cf,Cj) for the Bad-MO group) and p53 mutant lines (panels (Cc,Cg,Ck,Cm) for the Bad-M5 group; panels (Cd,Ch,Cl,Cn) for the Bad-MO group) at 48 hpf during Bad knockdown. Green fluorescence intensity was recorded and is shown in (Co). Scale bar = 200 µm. (D) Brain development was probed by used the marker gene fabp7a via in situ hybridization in the wild-type and p53 mutant lines. Changes in fabp7a marker gene expression at different angles were counted and are shown in (Df). Scale bar = 100 µm. All N = 7. All data were analyzed using either paired or unpaired Student’s t-tests, as appropriate. * p < 0.01; ** p < 0.02.

Fig 5: p53R248Q resulted in regulation of p-AKT. (A) The level of p-AKT was increased after p53R248Q overexpression in OVCAR3 cells. It should be noted that OVCAR3 cells were transiently transfected with either empty plasmid (1 µg/mL), wtp53 (1 µg/mL), or p53R248Q (1 µg/mL) for 24 h and then stimulated with EGF for 15 min. GAPDH expression was measured to serve as a loading control. (B) The band intensity of the immunoblots was quantified using ImageJ software. The relative intensities of p-AKT expression were normalized with the control group. (wtp53: wild-type p53 plasmid; p53R248Q: p53R248Q mutation plasmid).