Fig 2: Determination of the binding domains of PCV3 Cap with NPM1. (A) Engineering of truncated constructs of PCV3 Cap, GFP-Cap (1–38), GFP-Cap (39–106), GFP-Cap (107–174), and GFP-Cap (175–214). (B) The interaction regions between NPM1 and Cap confirmed by Co-IP in HEK293FT cells with co-transfection. At 24 h post-transfection, the cell lysates were immunoprecipitated with Pierce Anti-HA Magnetic Beads, western blot analysis using antibodies to HA, GFP, and ß-actin. (C) Colocalization analysis. PK15 cells were co-transfected with GFP-Cap, truncation constructs of GFP-Cap, and HA-NPM1. The cells were incubated with the antibodies corresponding to the HA tag at 24 h post-transfection, and followed by the Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with DAPI (blue).

Fig 3: NPM1, HSPE1, and HSPB1 were both O-GlcNAc-modified and phospho-modified. a ETD mass spectra of peptides derived from NPM1, HSPE1, and HSPB1. b The O-GlcNAcylation and phosphorylation levels of endogenous NPM1, HSPE1, and HSPB1 were measured in HB tissues and normal tissues by immunoprecipitation and western blot analysis. All experiments were repeated three times

Fig 4: Endogenous colocalization analysis. PCV3 Cap induced the translocation of NPM1 to the cytoplasm from the nucleus. (A,B) PK15 cells were transfected to express FLAG-Cap and GFP-Cap. The cells were incubated with the antibodies corresponding FLAG tag, NPM1, and NCL followed by the Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with DAPI (blue). (C) The cell nuclear and cytoplasmic extraction was performed after PK15 cells transfected GFP-Cap plasmid. At 48 h post-transfection, the protein samples were prepared and detected using antibodies against GFP and NPM1 by western blot analysis. Histone H3 and ß-actin were used as fractionation quality controls. (D) The relative gray intensity of cytoplasmic NPM1 was calculated using ImageJ. Two-tailed Student’s t-test was used for statistical analysis. Each experiment was independently done three times. Data represent by mean ± standard deviation (SD). Asterisks (*) indicate the statistical significance: *P < 0.05; NS: not significant.

Fig 5: Schematic diagram of the mechanism of TP-induced lung cancer cell inhibition via ribosomal stress initiation. TP induces nucleolus degradation and inhibits 45S rRNA synthesis by reducing the binding of Pol I and UBF to the rDNA promoter. The 18S rRNA degraded by TP disrupts ribosomal synthesis, resulting in the liberation of RPL23. The combination of RPL23 with MDM2 activates p53, which drives apoptosis, growth inhibition and cell cycle arrest. TP, triptolide; Pol I, RNA polymerase I; UBF, upstream binding factor; RPL23, ribosomal protein L23; MDM2, mouse double minute 2; NCL, nucleolin; B23, nucleophosmin; PUMA, p53-upregulated modulator of apoptosis; C, cleaved; p, phosphorylated; r, ribosomal.