Fig. 3: Cytosolic expression of mutp53(E285K) protein is independent of putative K285 ubiquitination.

A The effect of the E285K mutation on 281–294 helix stability was validated compared to wtp53 using ESMFold model. Red color indicates reduction in protein stability according to changes in pLDDT score from red (destabilizing) to cyan (stabilizing). Mutp53(E285K) is shown in red. Magnifications illustrate the molecular interaction network involving E285 (left) versus K285 (right). The key residues involved in this network: K132, E271, K164, and R273 are shown in green. The structure is colored according to changes in protein stability due to mutation as estimated by the ESMFold ΔpLDDT score from red (destabilizing) to cyan (stabilizing). B M#31 cells were pretreated or not with MG132 (10 µM) for 2 h and a TUBE assay performed. C M#31 cells silenced for endogens mutp53(E285K) were reconstituted with wtp53, mutp53(E285K), and mutp53(E285R), respectively, and cell death (PI⁺) monitored 24 h after stimulation with cisplatin (15 µM) using IncuCyte® technology (n = 3; mean ± SD; **p ≤ 0.01; ns not significant). D Prediction of the effect of K132E mutation on the p53 DBD structure using ESMFold model. The structure is colored according to changes in protein stability due to mutation as estimated by the ESMFold ΔpLDDT score from red (destabilizing) to cyan (stabilizing). K132E and E285K mutations are depicted in yellow and red, respectively. The other residues involved in the salt-bridge network formation (K132, E271, K164, and R273) are shown in green. E M#31 and M#54 cells stably silenced for endogenous p53 were reconstituted with wtp53, and mutp53 variants E285K, K132E, R175H, and R248W, respectively, and cell death (PI⁺) monitored 24 h after stimulation with cisplatin (15 µM) (n = 3; mean ± SD; **p ≤ 0.01; ns not significant). F The same M#31 transfectants were used to assess p21 and MDM2 expression by Western-blot analysis. β-actin served as loading control. G M#31 (E285K) and M#54 (wt) cells were stimulated with cisplatin (15 µM). After 24 h localization of MDM2, p53, p-p53(Ser15), and p21 was monitored in whole cell lysates as well as in cytosolic and nuclear fractions by Western-blot analysis. IκBα and histone H3 served as loading/fractionation controls.