Fig 2: Dox induces nucleolar stress in hCmPCs in absence of ROS, apoptosis and cytotoxicity. a hCmPCs treated with 1 μM Dox for 4 h and 8 h (right upper and lower panels, respectively) or DMSO as control (w/o Dox; left upper and lower panels). Representative images of immunofluorescence of NPM (green) and nuclei counterstained with DAPI (blue). NPM is localised within nucleoli in control-treated cells and delocalises into the nucleoplasm upon Dox treatment. Scale bar 10μm. b Dox inhibits new rRNA synthesis in hCmPCs. Pre-rRNA 45S levels were measured by qRT-PCR and normalised to 18S expression (n = 4 for each group *P < 0.05; ***P < 0.001 vs Ctrl w/o Dox t=0h). c Representative image of DCF FACS measurements of hCmPCs treated or not with 1μM Dox for 8h. Serum starved hCmPCs were treated with 1 μM Dox for 8h. After treatment, cells were treated with DCF for ROS detection. d Bar graph representing mean fluorescence intensity (MFI) of DCF. Dox treatment does not significantly modulate ROS compared to untreated cells (n = 4 for each group). e Representative image of Annexin V FACS measurements of hCmPCs treated or not with 1μM Dox for 8, 24, 48, and 72h. After treatment, cells were stained with Annexin V for apoptosis detection. 30 min of 10mM H2O2 was used as positive control. Dox treatment significantly induced apoptosis at 48h and 72h compared Ctrl cells (n = 4 for each group; *P < 0.05 vs t=0h). f Representative image of cytotoxicity in Dox-treated hCmPCs. hCmPCs were treated with 1 μM Dox and analysed for cytotoxicity at different time points described in the figure. hCmPCs exhibited a significant cell toxicity only at 48h of treatment with Dox (n = 6; *P < 0.05)

Fig 3: Dox-induced DSBs elicit a rapid NPM secretion in the absence of necrosis. a Representative WB using γH2AX and NPM antibodies showed that γH2AX levels were induced upon 1μM Dox in hCmPCs after 4 and 8h of treatment while NPM levels were not modulated. Beta-actin was used as loading control. b Densitometric analysis of γH2AX expression levels normalised by β-actin (n = 3;**P < 0.01;***P < 0.001). c Densitometric analysis of NPM expression levels normalised by β-actin (n = 3). Uncropped images of blots are shown in Additional file 1: Figure S1b. d hCmPCs were treated for 4 h and 8 h with 1 μM Dox, serum-free media were collected and analysed by ELISA assay for NPM expression. NPM was induced at 8h with respect to 4h treatment both in Ctrl- and Dox-treated cells. The difference at 8h between Dox and Ctrl was statistically significant (n = 7;*P < 0.05). e LDH assay, a marker of cell necrosis, showed that the release does not depend on a passive release from dead cells (n = 6). A negative control and positive control were inserted in the assay; the latter is statistically significant vs negative control (n = 6; *P < 0.05)

Fig 4: eNPM is rapidly secreted in the extracellular space by HaCaT, KCs, and HFs upon different inflammatory stimuli. (A) HaCaT were either serum starved for 8 h or treated with 100 μg/ml LPS, or 0.5 μg of Poly I:C (HMW e LMW), or with a MIX of cytokines (IL-17A, IL-22, TNFα, and IFNγ). Extracellular NPM levels (eNPM) were quantified by ELISA assay (n = 5; **p < 0.01, ***p < 0.001). All the treatments induced an increase eNPM compared to starvation. (B) HFs of healthy subjects were either serum starved for 8 h, or treated with 100 μg/ml LPS, or 0.5 μg of Poly I:C (HMW e LMW), or with a MIX of cytokines (IL-17A, IL-22, TNF-α, and IFN-γ). eNPM levels were quantified by ELISA assay (n = 8; *p < 0.05, **p < 0.01). Poly I:C and cytokine MIX increased eNPM significantly in the supernatants. (C) Healthy KCs and KCs of NLS of Pso were either serum starved or treated with a MIX of cytokines for 8 h. eNPM was quantified using a specific ELISA assay (n = 6; *p < 0.05, **p < 0.01). eNPM was increased in the supernatants of KCs isolated from NLS of Pso and in healthy KCs treated with the cytokine MIX compared to serum-starved healthy KCs supernatants. eNPM was also increased in the supernatants of KCs of Pso treated with cytokine MIX compared to serum-starved KCs of NLS of Pso supernatants.

Fig 5: Healthy donors and lung cancer patients have a repertoire of CD4 T cells that recognize citrullinated NPM. CFSE loaded PBMCs were stimulated with NPM266-285cit and proliferation was assessed on day 7–10. Responses double of medium control were considered to be significant. (A) FACs plot depicting representative CD4 and CD8 proliferation response to NPM266-285cit. Bar chart shows proliferation ratio of control in day 7 and 10 in (B) healthy donors and in (C) Lung cancer patients. (D) PBMCs were stimulated with NPM266-285cit with or without CD45RO depletion and responses were measured on days 7–11. Representative plots are shown. Proliferating CD4 T cells were phenotyped using (E(i)) IFNγ, (F(ii)) Granzyme B and (G(iii)) CD134 marker on day 10. NPM, nucleophosmin; PBMCs, peripheral blood mononuclear cells; CFSE, carboxyfluorescein succinimidyl ester.