Fig 2: Functional validation of N4BP1 in epithelial barrier maintenance and neutrophil signaling. (A) Interaction intensity histogram between epithelial cells and neutrophils in PD and PDT groups, analyzed by CellPhoneDB. Signaling pathways are color-coded by family: ANNEXIN (green), CXCL (red), LAMININ (blue), and SAA (purple). Left: PD group; Right: PDT group. (B) Box plots showing specific ligand-receptor pair probabilities across HC, PD and PDT groups. Communication probabilities for CXCL1/6-CXCR1/2 and ANXA1-FPR1/2 axes are significantly elevated in PD compared to HC, with partial attenuation in PDT. Each dot represents an individual sample; box boundaries indicate 25th and 75th percentiles; horizontal lines denote medians. (C) Sensitivity analysis of communication probability for ANNEXIN (left) and CXCL (right) pathways across HC, PD, and PDT groups. Box plots illustrate aggregated pathway-level communication probabilities derived from CellChat analysis. (D, E) N4BP1 knockdown in primary human gingival epithelial cells (PGECs). (D) Representative Western blots showing N4BP1, tight junction protein occludin (OCLN), keratin 5 (KRT5), and β-actin loading control following siRNA-mediated N4BP1 silencing. (E) Quantification of N4BP1 protein levels normalized to β-actin. Data represent mean ± SEM from three independent experiments; ***P < 0.001 by unpaired two-tailed Student’s t-test. (F, G) Pharmacological upregulation of N4BP1 in primary human neutrophils. Cells were treated with the proteasome inhibitor MG-132 (20 μM, 3 h) to prevent N4BP1 degradation. (F) Western blot confirmation of elevated N4BP1 protein levels following MG-132 treatment. (G) Secreted ANXA1 concentration in culture supernatants measured by ELISA. N4BP1 stabilization significantly attenuates ANXA1 secretion. Data represent mean ± SEM from three independent donors; ***P < 0.001 by unpaired two-tailed Student’s t-test. NC, negative control (scrambled siRNA for (E, F); DMSO vehicle for (F, G)). (H) Violin plots depicting expression levels of CXCL1 (left) and CXCL6 (right) across cell populations. N4BP1+ epithelial cells and neutrophils exhibit attenuated chemokine expression compared to N4BP1- counterparts, suggesting N4BP1-mediated suppression of pro-inflammatory chemokine production.