Fig. 7: NCF4 promotes the activation of NK and CD8⁺ T cells within the colonic microenvironment in the precancerous stage.

Single-cell RNA sequencing analysis of colonic cells isolated from WT (n = 3) and Ncf4^–/–mice (n = 3) at Day 35 post AOM treatment. a Uniform manifold approximation and projection (UMAP) plot and fraction illustrating the distribution of immune and epithelial cells colored by cluster. TA, transit-amplifying cells; Lig Enterocytes, large intestine gland enterocytes; ASC, adenoma-specific cells; EEC, enteroendocrine cells; and SeEPC, stem-early enterocyte precursor cells. b UMAP embedding of all epithelial cells with the stochastic representation of the RNA velocity. c UMAP plot of epithelial cells showing expression of a gene with stem-like features (tumor marker), Slc12a2 and Fermt1. d Representative immunohistochemical image of colon tissues from WT (n = 3) and Ncf4^–/– mice (n = 3) stained with anti-SLC12A2 and anti-FERMT1. Scale bars: 10 μm. e UMAP embedding of sub-clustering of CD4⁺ T, CD8⁺ T, and NK cell clusters, colored by subclusters. The identity of each cluster was redetermined by its predominant cells. The annotation of each subcluster was determined by its marker genes. f UMAP plot and fraction illustrating the distribution of the subcluster of immune cells in (e). The cells in the UMAP plot were colored by sample identity. g Chord diagram showing potential interactions or communication between different cell types mediated by IFN-γ signaling pathway. There was no significant interaction in Ncf4^–/– mice compared to the WT mice. TA transit-amplifying cells, TC TA carcinoma, DE1 distal enterocytes 1, LE large intestine gland enterocytes, AC adenoma-specific cells, EEC enteroendocrine cells. h, i Representative flow cytometry plots (h) and quantification analysis (i) of IFN-γ⁺ cells in colonic CD45.2⁺, CD4⁺ T, CD8⁺ T and NK cells from WT (n = 7) and Ncf4^–/– (n = 7) mice as indicated. Data are representative of 3 independent experiments with similar results (d, h, i). Data represent Mean ± SEM for (i), 2-sided Student’s t-test without multiple-comparisons correction, p-value is indicated in the graph. Source data are provided as a Source Data file.