Fig. 5: Multi-omics analysis reveals Il7r-KO-driven transcriptional and proteomic reprogramming via NF-κB/CXCL1 axis.

A Volcano plot of differentially expressed genes (DEGs) in IL7R-KO versus IL7R-WT cells (N = 3 vs. 3). Downregulated and upregulated genes are highlighted in blue and red, respectively; B heatmap of 30 significantly altered proteins identified by deep proteomics analysis, showing hierarchical clustering of expression patterns (N = 3 vs. 3); C Venn diagram depicting overlap between RNA-seq-derived DEGs and proteomics-identified differentially expressed proteins, highlighting concordant molecular changes at the transcriptomic and proteomic levels; D multiplex immunofluorescence of tumor tissues stained for PANCK (green, epithelial cells), CXCL1 (yellow), and DAPI (blue, nuclei) (N = 3). Representative ×20 images (scale bar: 20 µm) show reduced CXCL1 expression in IL7R-KO tumor sections compared to WT controls; KEGG pathway enrichment (E) and GSEA (F) analyses of DEGs in IL7R-KO cells, identifying NF-κB signaling and cytokine-related pathways as top functional categories; G CUT&Tag profiling of NF-κB binding sites in ID8-WT cells (N = 2). Density heatmap illustrates NF-κB occupancy within ±3.0 kb of transcription start sites (TSS). Representative genomic tracks (bottom) confirm NF-κB binding at the CXCL1 locus (blue boxes denote peak regions); Western blot analysis of total NF-κB, phospho-NF-κB (p-p65), and CXCL1 protein levels in: H IL7R-WT (N = 3) cells treated with NF-κB inhibitor (15 µM, 24 h) versus vehicle control (N = 3); I IL7R-KO versus IL7R-WT cells. GAPDH served as a loading control; J IF staining for p65 nuclear translocation (arrows indicate cells with nuclear p65 localization) (N = 3). Images acquired at ×20 magnification (scale bar: 100 µm); K flow cytometry analysis of immunosuppressive macrophage polarization in bone marrow-derived macrophages (BMDMs) co-cultured with murine Il7r-KO ovarian cancer cells treated with or without CXCL1 and Il7r-WT tumor cells (N = 3); L qPCR validation of macrophage polarization marker genes in BMDMs co-cultured with CXCL1-treated Il7r-KO tumor cells, Il7r-KO tumor cells and Il7r-WT tumor cells (N = 3); M Transwell migration assay to assess chemotaxis of BMDMs toward conditioned media from CXCL1-treated or untreated murine Il7r-KO ovarian cancer cells and Il7r-WT tumor cells. Migrated cells were quantified after 24 h of incubation (N = 3). Scale bar: 100 µm. Statistical significance: ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± SEM.