Fig. 5
Mettl3-induced m6A modification is involved in the upregulation of CBX8. a, RIP-qPCR showing the stronger enrichment of m6A in DLD-1 and SW480 cells than that in NCM460 cells. b, Mettl3 expression was assessed using data from the TCGA. C, CBX8 expression was positively correlated with Mettl3 expression in an analysis of TCGA data. d-e, Western blot of Mettl3 and CBX8 after Mettl3 inhibition or overexpression in DLD-1 cells. f, RIP-qPCR showing the enrichment of m6A in DLD-1 after Mettl3 depletion. g, The decay rate of CBX8 mRNA after treatment with 2.5 μM actinomycin D for indicated times, with Mettl3 knockdown or overexpression in DLD-1 cells. h, RIP-qPCR showing the enrichment of IGF2BP1 on CBX8 mRNA in DLD-1 and SW480 cells. i, Western blot of IGF2BP1 and CBX8 after IGF2BP1 inhibition in DLD-1 cells. j, The decay rate of CBX8 mRNA after treatment with 2.5 μM actinomycin D for indicated times, with IGF2BP1 knockdown in DLD-1 cells. k, RIP-qPCR showing the enrichment of IGF2BP1 on CBX8 mRNA in DLD-1 with Mettl3 knockdown. l, The schematic figure shows that mettl3 upregulates m6A level of CBX8 mRNA, IGF2BP1 binding to m6A sites to sustain the stability of CBX8 mRNA; CBX8 overexpression recruits KMT2b and Pol II to activate LGR5 by inducing H3K4me3, which regulates cancer stemness and chemosensitivity in CC. The relative enrichment in the RIP-qPCR assay was normalized by input. Data are shown as the mean ± SD of three replicates (*, P < 0.05; **, P < 0.01)