Fig. 4
From: m6A-dependent glycolysis enhances colorectal cancer progression
Transcriptome-wide m6A-seq and RNA-seq assays identified potential targets of METTL3 in colorectal cancer. (a) The m6A dot blot assay of global m6A abundance in mRNA of HCT116 WT and METTL3-knockout cells. MB, methylene blue staining (as a loading control). (b) The global m6A levels in mRNA of HCT116 WT and METTL3- knockout cells were measured by the EpiQuik™ m6A RNA Methylation Quantification Kit, n = 3, nonparametric Mann–Whitney test. (c) LC-MS/MS quantification of the m6A/A ratio in poly(A) RNA in HCT116 WT and METTL3-KO cells. (d) HOMER motif analysis revealed the top consensus m6A motif in HCT116 cells. (e) Graphs of m6A peak distribution illustrating the proportion of common m6A peaks in the indicated regions in HCT116 cells. (f) Cumulative curves of m6A abundance (log2(m6A EF + 1)) in HCT116 WT and METTL3-KO cells. The abundance of m6A immunoprecipitation was normalized to input when calculating the enrichment fold. P value was calculated using two-sided Wilcoxon and Mann–Whitney test. EF, enrichment folds. (g) Circos plot showing the distribution of hypermethylated (hyper) and hypomethylated (hypo) m6A peaks in the human transcriptome of HCT116 METTL3-KO cells compared with WT cells. (h) Distribution of genes with a significant change in both m6A level and gene expression level in HCT116 METTL3-KO cells compared with WT cells. (i) The flow chart for selected candidate target genes of METTL3 in HCT116 METTL3-KO cells is shown. (j) The relative abundance of m6A sites along HK2 and SLC2A1 (GLUT1) mRNA in HCT116 WT and METTL3-KO cells, as detected by m6A-seq. The orange rectangles indicated that the m6A peaks had a significantly decreased abundance. (k) Agarose electrophoresis and real-time PCR analysis of MeRIP assays in CRC cells showing the direct binding between the m6A antibody and HK2 5’UTR, HK2 3’UTR and SLC2A1 (GLUT1) 3’UTR. (l) MeRIP-qPCR analysis of HK2 5’UTR, HK2 3’UTR and SLC2A1 (GLUT1) 3’UTR m6A levels in HCT116 WT and METTL3-KO cells, n = 3, nonparametric Mann–Whitney test. (WT, wild type; METTL3-KO, METTL3-knockout)