Figure 5. NPM1 dissociates from rRNA/rDNA following S-glutathionylation.

(a) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after RNase A (1 mg ml⁻¹) and DNase I (0.5 U ml⁻¹) digestions in HeLa cells with or without H₂O₂ (500 μM) treatment. (b,c) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP (b) and ChIP (c) assays in HEK293T cells treated with H₂O₂ (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t-test, **P<0.01 or ***P<0.001 with respect to treated versus untreated or −NAC versus +NAC cells. (d,e) Similar experiments to b,c; the relative quantities of rRNA (d) and rDNA (e) bound with FLAG-NPM1 WT or mutant C275S in HEK293T cells exposed to H₂O₂ (500 μM) were assessed. **P<0.01 or showed above the bars. In b–e, Mock referred to the transfection negative control, whereas Vehicle referred to the treatment negative control. Data were represented as mean±s.e.m. Scale bars, 5 μm. The internal controls of RIP and ChIP assays are shown in Supplementary Fig. 5b–d.