Fig. 4From: RETRACTED ARTICLE: m6A modification-mediated CBX8 induction regulates stemness and chemosensitivity of colon cancer via upregulation of LGR5CBX8 maintains the H3K4me3 status at the LGR5 promoter by binding to KMT2b. a, Overview of the LGR5 promoter region with ChIP-seq data from ENCODE for H3K27me3, H3K4me3 and H3K27Ac in HCT-116 cells and for KMT2b in HepG2 cells. b, ChIP-qPCR analysis identified enrichment of H3K3me3 in the CBS1-CBS4 regions after CBX8 knockdown in DLD-1 cells. ChIP-qPCR with IgG was performed as the control. c, ChIP-qPCR analysis identified enrichment of H3K3me3 in the CBS1-CBS4 regions after CBX8 overexpression in DLD-1 cells. ChIP-qPCR with IgG was performed as the control. d, The RNAi screen of KMT2s (KMT2A-G) showed that KMT2b siRNA significantly downregulated LGR5 mRNA levels in DLD-1 cells. e, Western blot analysis of LGR5 expression in KMT2b knockdown DLD-1 cells overexpressing CBX8. f, CoIP showed the interaction between the CBX8 and KMT2b proteins in DLD-1 cells. g, HEK293 cells were transfected with Flag-tagged CBX8 with or without HA-tagged KMT2b. Lysates were subjected to IP using anti-Flag and anti-HA antibodies. h, ChIP-qPCR analysis performed to determine the binding affinity of KMT2b to the seven LGR5 promoter regions in DLD-1 cells showed that CBX8 bound to the KBS1-KBS3 regions in the LGR5 promoter. ChIP-qPCR with IgG was performed as the control. i, ChIP-qPCR analysis was used to determine the binding affinity of KMT2b to the KBS1-KBS3 regions after CBX8 knockdown. j, ChIP-qPCR analysis showed the enrichment of CBX8 and H3K4me3 in the CBS1-CBS3 regions after KMT2b knockdown in DLD-1 cells. k, Schematics of the luciferase reporter gene constructs. l, The luciferase reporter gene constructs were cotransfected with shCBX8 or shCtrl into DLD-1 cells, and reporter gene activity was measured after 48 h by a dual luciferase assay. The relative value in DLD-1 cells cotransfected with pGL3 (− 1224/317) and shCtrl was set to 100%. m, The luciferase reporter gene constructs were cotransfected with the CBX8 overexpression plasmid or empty vector into DLD-1 cells, and reporter gene activity was measured after 48 h by dual luciferase assay. n, Schematic of the five Flag-CBX8 recombinant proteins (CB1-CB5). o, Plasmids encoding a Flag-tagged, CBX8 truncation mutant were transfected into DLD-1 cells, anti-Flag antibody was used to immunoprecipitate the bound proteins, and the KMT2b level in the immunoprecipitates was determined by Western blot. Data are shown as the mean ± SD of three replicates (*, P < 0.05; **, P < 0.01)Back to article page