Fig 1: Ppia was mono-ADP ribosylated at the E140 residue by PARP3 to promote macrophage inflammation. A Schematic diagram of the homology of Ppia in mice, rats and humans. B Schematic diagram of the Ppia mutation sites. C Comparison of the effects of the D9A, E120A and E140A mutants on promoting inflammation (n = 3). The mRNA levels of IL-1β, IL-6 and Ppia were detected by qPCR. D RAW264.7 cells were transfected with the Ppia-WT and Ppia-E140A plasmids, and their ability to stimulate inflammation was tested by determining the phosphorylation status of p65 (n = 3). Protein levels of phosphorylated p65 were measured by western blot. E The modification ability of the purified Ppia-WT and Ppia-E140A mutant proteins in the in vitro system was tested by western blot (n = 3). F Mono-ADP-ribosylation on Ppia E140 was confirmed by immunoprecipitation of Ppia and detection with a mono-ADPr antibody (n = 3). G The purified Ppia-WT and Ppia-E140A proteins were added to RAW264.7 cell cultures to detect the expression of IL-1β and IL-6 by qPCR and (H) phosphorylation of p65 by western blot (n = 3). I The secretion of Ppia was influenced by mono-ADP ribosylation and was detected by ELISA (n = 7). J The purified Ppia-WT and Ppia-E140A proteins were added to MPM cell cultures to detect the expression of IL-1β and IL-6 by qPCR (n = 3). K Schematic diagram of the mechanism by which PAPR3 mono-ADP ribosylate Ppia to stimulate the NF-κB pathway and activate inflammation in macrophages. The data were expressed as the means ± standard deviations. One-way ANOVA was used for statistical analysis in (C-J). *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001 were considered significant
Fig 2: Ppia mediated PARP3 to promote inflammation. A The proinflammatory effect of PARP3 requires the mediation of Ppia. The expression of IL-1β was inhibited with the knockdown of Ppia (n = 3). B and the phosphorylation of p65 was also suppressed. (n = 3). C The presence of the PARP3 inhibitor ME0328 inhibited the increase in IL-6 and D the phosphorylation of p65 induced by Ppia overexpression (n = 3). E LPS-induced IL-1β & IL-6 expression (F) and p65 phosphorylation were inhibited by transfecting the siRNA of Ppia (n = 3). The mRNA levels of IL-1β, IL-6, PARP3 and Ppia were detected by qPCR and protein levels of phosphorylated p65 were measured by western blot. The data were expressed as the means ± standard deviations. One-way ANOVA was used for statistical analysis in (A-F). *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001 were considered significant
Fig 3: Ppia interacted with PARP3. A Schematic diagram of the screen for the candidate substrate of PARP3. B The top five proteins among the fifteen intersecting proteins were listed. These proteins were candidate substrates for PARP3. C The peptide map of the Ppia protein was presented. D Co-IP validated the interaction between PARP3 and Ppia
Fig 4: Transcriptomic analysis of eCypA-regulated genes in NALM6 cells.NALM6 cells were serum-starved for 6 h and then treated with IgG, CypA + IgG, or CypA + Anti-CypA with three biologically independent samples analyzed per group (n = 3 per group). Raw transcriptome reads were deposited in the China National Center for Bioinformation (BioProject accession: PRJCA035020; raw data accession: HRA010096). (A) Bar plot showing the numbers of upregulated and downregulated genes; 46 genes were upregulated by CypA and downregulated by Anti-CypA. (B, C) Volcano plots of differentially expressed genes generated from RNA-seq analysis. Differentially expressed genes were identified using a threshold of P < 0.05. (D) Gene Ontology (GO) enrichment analysis of differentially expressed genes between IgG and CypA + IgG-treated NALM6 cells. (E) KEGG pathway enrichment analysis of differentially expressed genes between CypA + IgG and CypA + anti-CypA-treated NALM6 cells. Source data are available online for this figure.
Fig 5: eCypA exacerbates ALL severity in a mouse model.(A) NALM6-Luc cells were injected via the tail vein into NCG mice, followed by weekly intraperitoneal administration of recombinant IgG (n = 3), CypA+IgG (n = 5), or anti-CypA monoclonal antibody (mAb) (n = 5) starting the next day. (B, C) ELISA analysis of mouse (B) and human (C) eCypA levels in peripheral blood (PB) plasma on day 18 after NALM6-Luc cell injection; samples from Ctrl mice (n = 3, no leukemia cell injection) were included as baseline controls. (D) Bioluminescence imaging of mice on day 21 following intraperitoneal injection of luciferin substrate (left), with quantitative analysis using an in vivo imaging system (right). Each data point represents the bioluminescence intensity from an individual mouse (IgG, n = 3; CypA + IgG, n = 5; Anti-CypA, n = 5). (E) Hematoxylin and eosin (H&E) staining and hCD19 immunohistochemistry (IHC) of spleen tissue sections collected on day 22 after leukemia cell injection (left), with quantification of hCD19⁺ areas using ImageJ (right). Scale bars, 200 and 50 μm as indicated. Each data point (n) represents one microscopic field, with four randomly selected fields analyzed per mouse (IgG, n = 12; CypA + IgG, n = 20; Anti-CypA, n = 20). (F) Schematic of the p190-BCR-ABL-driven B-ALL model in C57BL/6 mice. Primary leukemic spleen cells were harvested after 2 weeks of disease development and transplanted into secondary recipient mice via tail vein injection. Anti-CypA mAb treatment was initiated the next day and administered weekly for 3 weeks; mice were sacrificed on day 18 post-transplantation. (G) ELISA analysis of mouse eCypA levels in PB plasma from Ctrl (n = 4), IgG (n = 6), and Anti-CypA (n = 6) groups. (H) Flow cytometric analysis of GFP⁺ leukemic cell infiltration in bone marrow (BM) and spleen from IgG and Anti-CypA groups, including representative flow cytometry plots and quantification (n = 6). Data were presented as mean ± SD; n represents individual mice (B–D, G, H). In (E), n represents independently analyzed microscopic fields. Statistical significance was determined using one-way ANOVA for (B, C, G), unpaired two-tailed Student’s t-test for (D), and two-way ANOVA for (E, H). *p < 0.05, **p < 0.01, ***p < 0.001; ns not significant. Source data are available online for this figure.
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