Fig 1: Anti-DEFB1 monoclonal antibody as a promising therapeutic agent against lung adenocarcinoma.A, B High-content imaging for the proliferation of A549 DEFB1-NC/KO/ROE cells treated with 1 μg/mL IgG or anti-DEFB1 monoclonal antibody mAb-5 (scale bar: 200 μm); C Transwell assay for the invasion capacity of A549/PC-9 cells treated with 1 μg/mL IgG or mAb-5 (scale bar: 100 μm); D Western blot detection of the expression of epithelial-mesenchymal transition (EMT)-related proteins in A549/PC-9 cells treated with 1 μg/mL IgG or mAb-5; E Flow Cytometry for evaluating the M1/M2 polarization statues of THP-1 M0 cells co-cultured with A549/PC-9 cells treated with 1 μg/mL IgG or mAb-5; F–H In vivo proliferative capacity in nude mice CDX model and in human lung adenocarcinoma organoids (scale bar: 100 μm) treated with IgG or mAb-5 (60 μg per injection, every 3 days, for a total of 9 times); I Schematic description of KrasG12D-driven spontaneous lung tumor mice model expressing humanized DEFB1 and the treatment mode; J, K Representative images of chest computed tomography before and after the treatments of IgG or mAb-5 (60 μg per injection, every 3 days, for a total of 9 times) and survival analysis in spontaneous lung cancer model; L Epithelial-mesenchymal transition (EMT) and macrophage infiltrating statuses in spontaneous lung cancer model treated with IgG or mAb-5 (scale bar: 200 μm); M Mechanistic schema of DEFB1 in lung adenocarcinoma. Statistical analysis: B Two-way ANOVA test (n = 6) and Bonferroni correction; G Student’s t-test; K Log-rank test (n = 6).
Fig 2: DEFB1 exhibits elevated expression and indicates unfavorable survival in lung adenocarcinoma.A, B Intersection of genes that might promote lung adenocarcinoma proliferation obtained from in vitro and in vivo CRISPR/Cas9 genome-wide knockout experiments and genes associated with worse prognosis of lung adenocarcinoma; C, D UMAP clustering of single-cell transcriptome sequencing data for cell clustering and DEFB1 expression levels in each single cell; E, F DEFB1 expression in 118 pairs of lung adenocarcinoma tumor tissues and adjacent normal lung tissues by multiple immunohistochemical staining (red) (scale bar: 200 μm); G, H Western blot results of DEFB1 expression in normal lung epithelial cell lines, lung adenocarcinoma cell lines, as well as six pairs of tumor tissues and adjacent normal tissues obtained from patients with lung adenocarcinoma; I, J Kaplan–Meier and Cox survival analyses between tumor DEFB1 expression and progression-free survival in lung adenocarcinoma patients based on multiple immunohistochemical staining results. Statistical analysis: F Mann–Whitney U-test, and G Log-rank test.
Fig 3: DEFB1 interacts with macrophage Migration Inhibitory Factor (MIF) to promote M2 polarization of macrophages in vitro and in vivo.A Infiltrating status of M1 (marked by F4/80 and INOS) and M2 (marked by F4/80 and CD206) macrophages in nude-mouse xenografts derived from A549 DEFB1-NC/KO/ROE cells (scale bar: 200 μm); B CoIP verification of the interaction between DEFB1 and MIF in A549/PC-9 cells treated with exogenously added 3× Flag-tagged DEFB1 (200 pg/mL); C AlphaFold 3 prediction of the interaction mode between DEFB1 and MIF; D Schematic diagram of an in vitro co-culture model of lung adenocarcinoma cells and THP-1 derived macrophages; E, F Flow Cytometry and qRT-PCR for evaluating the effects of DEFB1 and/or MIF inhibitor (ISO-1) on M1 polarization of THP-1 M0 cells co-cultured with A549 cells; G, H Flow Cytometry and qRT-PCR for evaluating the effects of DEFB1 and/or ISO-1 on M2 polarization of THP-1 M0 cells co-cultured with A549 cells; I, J Effects of DEFB1-KO, PPL-KO, and/or ISO-1 on the in vivo proliferative capacity in nude-mouse CDX model. Statistical analysis: (F, H, and G) One-way ANOVA test and Bonferroni correction.
Fig 4: DEFB1 interacts with Periplakin (PPL) to promote epithelial-to-mesenchymal transition (EMT) and proliferation in lung adenocarcinoma cells.A, B Flowchart and results for screening interacting membrane-localized and secreted proteins using co-immunoprecipitation (CoIP) combined with mass spectrometry and anti-Flag in A549 cells treated with exogenously added 3× Flag-tagged DEFB1 (200 pg/mL); C CoIP verification of the interaction between DEFB1 and PPL in A549/PC-9 cells treated with exogenously added 3× Flag-tagged DEFB1 (200 pg/mL); D AlphaFold 3 prediction of the interaction mode between DEFB1 and PPL; E Immunofluorescence assay for detecting the cellular distribution of Flag-DEFB1, ATP1A1 (plasma membrane marker), and PPL in A549/PC-9 cells treated with exogenously added 3× Flag-tagged DEFB1 (200 pg/mL)(scale bar: 30 μm); White dashed lines indicate the regions used for fluorescence intensity profile analysis; F Epithelial-mesenchymal transition (EMT) status of nude-mouse xenografts derived from A549 DEFB1-NC/KO/ROE cells (scale bar: 100 μm); G Western blot detection of the expression of EMT-related proteins in A549 DEFB1-NC/KO/ROE/KO + EXO cells with or without PPL knockout (PPL-KO); H High-content imaging (HCI) for the proliferation of A549 DEFB1-NC, and DEFB1-NC/KO/ROE/KO + EXO cells with PPL-KO (scale bar: 200 μm); I, J Transwell assays of the migration and invasion capacities of A549 DEFB1-NC, and DEFB1-NC/KO/ROE/KO + EXO cells with PPL-KO (scale bar: 100 μm). Statistical analysis: J One-way ANOVA test and Bonferroni correction.
Fig 5: DEFB1 enhances lung adenocarcinoma proliferation, migration, and invasion in vitro and tumorigenesis in vivo.A, B Western blot and ELISA for DEFB1 protein expression in A549/PC-9 DEFB1-NC/KO/ROE cells; C, D High-content imaging (HCI) for the proliferation of A549/PC-9 DEFB1-NC/KO/ROE/KO + EXO cells (scale bar: 200 μm); E Transwell assay for the invasion capacity of A549/PC-9 DEFB1-NC/KO/ROE/KO + EXO cells (scale bar: 100 μm); F Schematic diagram of for the xenograft experimental process; G, H In vivo proliferation and final tumor weights of A549 DEFB1-NC/KO/ROE cells. Statistical analysis: B, H One-way ANOVA and Bonferroni correction; D Two-way ANOVA test (n = 6) and Bonferroni correction.
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