Fig 2: FCN1 is enriched in macrophages of PIBD colon mucosa and related to hyper-inflammation. A Diagram depicting the analysis scheme of public single-cell RNA sequencing data (GSE121380). B t-SNE plots of CD68 (macrophage marker), FCN1 and IL1B at the single-cell level. C The bar plot of significantly enriched GSEA Hallmark pathways in FCN1high macrophages compared with FCN1low macrophages. D The enrichment plot of TNFa Signaling via NF-?B pathway. E Heatmaps of FCN1 and the top 15 up-regulated genes of TNFa Signaling via NF-?B pathway in FCN1high compared with FCN1low macrophages. F The heatmap showing the overall outgoing signaling patterns of major cell types, including B cells, epithelial cells, FCN1low macrophages, FCN1high macrophages, fibroblasts, and T cells, as predicted by CellChat. G-J Heatmaps of differential signaling networks between FCN1low macrophages and FCN1high macrophages, including MIF signaling, ICAM signaling, CCL signaling, and BAFF signaling

Fig 3: Validation of the diagnostic efficacy of FCN1 in our patient cohort. A Immunohistochemistry staining showing increased infiltration of FCN1+ cells in colon biopsies of IBD compared with non-IBD subjects. B Immunofluorescence staining showing that FCN1+ cells (red) colocalize with CD68+ cells (green) in the colon biopsies of children with IBD. Yellow indicates double positive cells. C-F Relative mRNA expression levels of FCN1, LINC10558, S100A8, and S100A9 in PBMCs from our validation cohort were measured by qRT-PCR. Data represent mean ± SD. Unpaired t test was employed to calculate P values. *P < 0.05, ****P < 0.0001. G ROC curves and their corresponding AUCs of FCN1, LINC01558, S100A8, and S100A9 based on relative mRNA expression levels in PBMCs from our validation cohort. H Based on PBMCs isolated from whole blood, FCN1 showed superior sensitivity and specificity for discriminating PIBD patients from non-IBD controls in our validation cohort

Fig 4: FCNB expression is upregulated and colocalized with CD68 in murine colitis model. A Schematic of the animal experiment design. Four-week-old C57BL/6 mice were treated with 3% DSS in drinking water for 7 days to induce colitis, while the control group received water. On the 8th day, mice were sacrificed for further analysis. B Mice with DSS-induced colitis showed significantly decreased body weight compared with the control mice. C Colon lengths were measured 8 days after DSS treatment. D Gross pictures of spleens and spleen index (%). Spleen index (%) is the ratio of spleen weight to body weight. E Representative H&E-stained colon tissue sections of control and colitis groups. Scale bars, 50 µM. F The mRNA expression levels of Fcnb (the murine homologue gene of human FCN1) in peripheral whole blood from control and colitis groups were measured by qRT-PCR. The protein expression levels of FCNB and IL-1ß in colon G and spleen H were measured by Western blotting. ß-actin was used as a loading control. I Immunohistochemistry staining of FCNB in colon of mice. Scale bars, 20 µM. J Immunofluorescence staining showing that FCNB+ cells (red) colocalize with CD68+cells (green) in the colon from mice with DSS-induced colitis. Yellow indicates double positive cells. Scale bars, 20 µM. Data represent mean ± SD. Unpaired t test was used to calculate P values. *P < 0.05, ***P < 0.001, ****P < 0.0001

Fig 5: FCN1 is upregulated in PIBD and associated with immune response. A The expression level of FCN1 in the rectal biopsies from PIBD and non-IBD subjects of the GSE117993 RNA-seq dataset. The y axis represents log2-scaled normalized counts by applying the regularized log (rlog) algorithm in DESeq2. Upregulated FCN1 expression in the colon B and peripheral whole blood (C) from PIBD and non-IBD subjects of the GSE126124 microarray dataset. The y axis represents log2-scaled normalized gene expression by using the robust multichip average (RMA) algorithm. Unpaired t test was employed to calculate P values. D GO enrichment analysis of DEGs between PIBD and non-IBD subjects. The bar plot exhibits significantly enriched GO terms and the pie chart shows the frequency of FCN1 involved in top 100 enriched GO terms. E The bar plot of significantly enriched FCN1-related MeSH terms of DEGs in PIBD compared with non-IBD subjects. F The protein-protein interaction subnetwork of DEGs associated with FCN1 based on STRING database. G The heatmap visualizing the relative expression of FCN1-associated genes in the GSE117993 dataset. H ROC curves and their corresponding AUCs of FCN1, S100A8 and S100A9 (subunits of calprotectin) in the colon validation datasets