Fig. 6

Promoter methylation status of EN1 and its correlation with EN1 mRNA expression. A Target sequences in the EN1 promoter region. The genomic position of the amplified fragment was obtained from the University of California Santa Cruz genome browser using the human (GRCh38) assembly. F and R represent forward and reverse primers, respectively. CpG sites in primer sequences are highlighted in grey. B Sanger sequencing results. The sequence results for the fragment are shown on top. The arrows indicate converted C-T events. C Trendline of EN1 promoter region hypermethylation correlated with individual tumours. D EN1 methylation levels in SACC tumour and normal tissues. E Bioinformatic analysis of the correlation between EN1 methylation and mRNA expression in 22 SACC samples. An inverse correlation was identified between EN1 methylation and mRNA expression. F EN1 methylation levels in SACC-83 and SACC-LM cell lines. G–J SACC-83 and SACC-LM cells treated with or without 5-aza-dC were evaluated for EN1 methylation by qMSP (G, I) and EN1 mRNA levels by qRT-PCR (H, J). **P < 0.01, ***P < 0.001, by t-test