Fig. 6. Nfkbia is the direct target of m⁶A modification in colonic epithelial cells.

(A) UpSet plot showed genes overlapped between the RNA-seq data and m⁶A RIP sequencing datasets. DEGs, differentially expressed genes. (B) mRNA isolated from colonic epithelial cells was immunoprecipitated (IP) with an anti-m⁶A antibody, and qRT-PCR was used to assess the mRNA level of Nfkbia (n = 5 per group). (C) Colonic epithelial cells were treated with actinomycin D for 4 hours, and Nfkbia mRNA was normalized for 0 hours (n = 6 per group). (D) Violin plot showing the distribution of Nfkbia per single cell in each cluster. (E) Bubble heatmap showing expression of TNF signaling pathway–related genes for each cluster. (F) Representative immunoblot for NFKBIA, Bcl2, and Bax in colonic epithelial cells from 2-week-old mice; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (G) Crypts isolated from Mettl14 WT and Mettl14 KO mice were grown in 3D organoid culture medium with or without RIPK1 inhibitor (GSK’963, 1 μM) (n = 5 per group). Data are presented as means ± SD. Two-sided Student’s t test (B, C, and G) was performed (**P < 0.01).