Figure 1. RNF5 Is Crucial for the Maintenance of Intestinal Homeostasis.

(A) Representative immunoblot of RNF5 in IECs from different intestinal regions of WT and Rnf5^−/− mice: duodenum (D), jejunum (J), ileum (I), and colon (C).

(B) Representative IHC staining of RNF5 in small intestine and colon sections of WT and Rnf5^−/− mice. Scale bars, 50 μm.

(C) Representative H&E staining of WT and Rnf5^−/− colon sections (red triangles indicate infiltrating lymphocytes). Scale bars, 50 μm.

(D) Representative images of immunofluorescence staining of CD45 in WT and Rnf5^−/− colon sections and quantification of CD45⁺ cells (n = 10, two fields each). Scale bars, 50 μm.

(E) Flow cytometric analysis of the frequencies of CD4⁺, CD4⁺CD44⁺, CD4⁺CD25⁺, CD8⁺, and CD8⁺CD44⁺ T cells, colonic DCs (CD45⁺ CD11c⁺ CD103⁺ F4/80^-), colonic macrophages (CD45⁺ CD11c⁺ CD103^- F4/80⁺), inflammatory monocytes (CD45⁺ CD11c^- Ly6C⁺), neutrophils (CD45⁺ CD11c^- Ly6G⁺), and NK1.1⁺ NK cells among gated CD45⁺ cells isolated from the lamina propria of colons from WT and Rnf5^−/− mice (n = 6).

(F) (F) Mean fluorescence intensity (MFI) of MHC classes I and II on colonic DCs cells isolated from the lamina propria of colons from WT and Rnf5^−/− mice (n = 6). (G) qRT-PCR analysis of elative mRNA expression of the indicated cytokines in WT and Rnf5^−/− colon tissues (n = 6).

(G) (H) Heatmap analysis of RNA-seq data performed from three naive WT or Rnf5^−/− colon tissues per group (left). One hundred fifty-six significantly dysregulated genes were identified in Rnf5^−/− colon tissues compared with naive Rnf5^−/− colon tissues (false discovery rate [FDR] adjusted p value < 0.05 by Benjamini-Hochberg method). Right table shows the top 10 upregulated genes identified in Rnf5^−/− colon tissues.

(H) All data are representative of three independent experiments. Graphs show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-tailed t test (D–G).