Figure 1.

DNMT1 suppressed Nanog expression by methylating the Nanog promoter in tumor cells. A) BSP analysis showing different methylation patterns of the Nanog promoter between CSCs and non‐CSCs. Ten clones were sequenced for each CG in the Nanog promoter. B) FACS analysis of the GFP⁺ cell population in tumor cells transfected with plasmids harboring a Nanog promoter with or without a single nucleotide mutation at CG4 or/and CG5. C) DNMT (DNMT1, DNMT3A, and DNMT3B) expression in CSCs and non‐CSCs analyzed by qRT‐PCR and WB in triplicate. D,E) qRT‐PCR and WB analysis of Nanog expression in tumor cells with D) DNMT1 upregulation and E) DNMT1 downregulation in triplicate. F,G) Methylation pattern of the Nanog promoter in tumor cells with F) DNMT1 upregulation and G) DNMT1 downregulation assessed by BSP analysis. Ten clones were sequenced for each CG in the Nanog promoter. B–E) Representative data of triplicate experiments are shown as the mean ± standard deviation (SD). A,F,G) P value was assessed by Fisher's exact test. C–E) One‐way ANOVA with Dunnett‐t test in comparison with WT, or Student's t test. NS, no significant difference. Representative images of triplicate WB experiments are shown.