Fig. 3

Validation of methylation status of candidates that were undergone DNA methylation and expression changes following G9A knockdown and their biological roles and clinical significance in NSCLC. a Methylation status of FOXP1 in TICs (LCSC1 and LCSC4) following G9A knockdown compared to their control as determined by Bisulphite PCR Sequencing (BS), chromatograms for the representative CpG dimers, and the methylation status (the red circles represent for methylated and the green circles represent for unmethylated CpG dimers) of the FOXP1 gene CpG dimers in TICs. The upper panel of chromatogram is for cancer cells transfected with control shRNA, the middle and lower panels are for TICs transfected with G9a shRNA. b Chromatogram of BS (as a representative example). c The protein encoded by genes methylated and silenced by G9A are upregulated following G9A knockdown (left panel) and treatment of TICs by G9A inhibitor UNC0642 resulted in increased expression of these proteins (CDYL2, DPP4, SP5, STAMBPL1, FOXP1 and ROBO1). d Biological processes and pathways which are generated from the genes that are upregulated following G9A knockdown. Three genes FOXP1, DPP4, and ROBO1 are associated with biological processes/pathways as shown. e Kaplan-Meier survival analysis of mRNA expression data of lung cancer indicates that high-expression of six candidate genes (CDYL2, DPP4, SP5, STAMBPL1, FOXP1, ROBO1) combined or f each individual gene (e.g., FOXP1) correlates to better clinical outcomes of patients in cancers (For both e and f, n = number of patients whose mRNA for respective genes were used for Kaplan-Meier analyses)