Figure 4: NPM1 is S-glutathionylated at Cys²⁷⁵ under stress.

(a) Non-reduced co-IP assay. After gradient doses of H₂O₂ (500 μM, 1 mM) treatments±antioxidant NAC (5 mM) pretreatment, S-glutathionylated NPM1 in HeLa cells was co-immunoprecipitated and analysed with either anti-GSH or anti-NPM1 antibodies. (b) Tandem mass spectrometry (MS/MS) analysis of NPM1 protein derived from non-reduced co-IP. FLAG-NPM1 was transfected to HEK293T cells exposed to H₂O₂ (500 μM) and immunoprecipitated by anti-FLAG M2 gel. The amino acid sequence of peptides was reconstructed by analysis of the b^−ion or y^−ion series. The MS/MS spectrum of the m/z y₅⁺ (972.4026) was corresponding to the NPM1 peptide KNCFR S-glutathionylated at Cys²⁷⁵, in comparison with the non-modified spectrum 667.3344 in Supplementary Fig. 4c. (c) NPM1 translocation (left) and RFI changes of nucleolar NPM1 (right, n=57 cells) after H₂O₂ (500 μM) treatment in HeLa cells. GSTP were silenced with two siRNA oligo and the relative GSTP mRNA level were showed (middle, siGSTP-1 and siGSTP-2). Unpaired t-test, **P<0.01 or ***P<0.001. (d) Translocation of EGFP-NPM1 WT and C275S mutant fused with active or inactive Grx1 at N-terminal upon H₂O₂ (500 μM) exposure. (e) The surface representation of NPM1 binding interface was based on NPM1-C70 structure (PDB 2vxd) with Cys²⁷⁵ coloured in magenta. GSH molecule, shown in sphere with nitrogen atom in blue, oxygen in red and carbon atom in green, could covalently link to Cys²⁷⁵ through thiol exchange. Scale bars, 10 μm (c) and 5 μm (d). Data were represented as mean±s.e.m. Grx1(ss), inactive Grx1. NPM1-SG, S-glutathionylated NPM1. NPM1-C70, C-terminal of NPM1 (70 amino acids).