Figure 7. NPM1 translocation is required for p53 activation.

(a,b) Endogenous NPM1 translocation and p53 accumulation cells under nucleolar stress with western blot validation. (a) Cells were pretreated with NAC (5 mM) for 4 h before Act.D (8 nM) treatment, or co-treated with DTT (5 mM) and Act.D (8 nM). Nucleolar line profiles of NPM1 in representative cells are shown in Supplementary Fig. 6a. (b) NPM1 was silenced with shRNA (shNPM1 5′-untranslated region (UTR)) before Act.D treatment or H₂O₂ (500 μM). (c) Immunofluorescence for endogenous p53 accumulation in NPM1-silenced cells (shNPM1 5′-UTR) that added back FLAG-NPM1 WT, C275S or WW mutants before treatment with Act.D. Line profiles of p53 in representative cells were showed in Supplementary Fig. 6c. (d) p53 accumulation after Act.D (left) or H₂O₂ (right) treatment in NPM1-silenced cells which were added back WT or C275S mutant of NPM1. Endogenous and exogenous FLAG-NPM1 were blotted with anti-NPM1 antibody. Nutlin-3 was used as a positive control for p53 inducer. (e) Relative mRNA level changes of p21 and PUMA after Act.D treatments in NPM1-silenced cells (shNPM1 5′-UTR) that added back FLAG-NPM1 WT or C275S mutants. Unpaired t-test. *P<0.05 or showed above the bars. Data were represented as mean±s.e.m. Scale bars, 10 μm. WW, W280/290A. All of the experiments were performed in U2OS cells.