Figure 6.

Identified Hits Do Not Lead to Structural Mutant p53 Reactivation

(A) U2OS (p53^WT) cells containing the p53 activity reporter show nuclear accumulation of p53 upon 10 μM Nutlin-3 treatment and induction of the activity reporter.

(B) Cal-33 (p53^R175H) cells containing the p53 activity reporter show nuclear accumulation of p53^R175H and no p53 activity reporter induction after Nutlin-3 treatment (data not shown) but induction after transduction of a CMV-driven p53 expression vector.

(C) Saos2 (p53−/−) cells containing the p53 activity reporter showed no staining for p53 and no induction of the p53 activity reporter after Nutlin-3 treatment (data not shown) but induction of the p53 activity reporter after transduction of a CMV-driven p53 expression vector. Luminescent readout normalized to DMSO controls. Exemplary data of multiple experiments are shown (n ≥ 2) Scale bars, 10 μm.

(D) p53 activity reporter activation upon aminothiazole treatment in cells with different p53 mutation status. Luminescent readout normalized to DMSO controls. Exemplary data of multiple experiments are shown (n ≥ 2), Bars show mean with SD (n = 8).

(E) Expression of structural mutant p53 in p53^−/− cells does not enhance p53 activity reporter activation. Luminescent readout normalized to DMSO controls. Exemplary data of multiple experiments are shown (n ≥ 2), Bars show mean with SD (n = 8).

(F) p53 knockdown validated by IF and western blot; partial p73 knockdown shown in western blot. Exemplary data of multiple experiments are shown (n ≥ 2); full western blot data show in Figure S6. Scale bar, 10 μm.

(G) p53 knockdown does not prevent p53 activity reporter activation in Cal-33 cells, whereas p73 knockdown shows suppression of p53 activity reporter activation. Luminescent readout normalized to DMSO controls. Exemplary data of multiple experiments are shown (n ≥ 2), Bars show mean with SD (n = 10). ∗∗∗p value < 0.001; ns, not significant.