Fig. 2.

Expression and methylation analyses of SPAG6 and L1TD1 in NHBECS and in NSCLC cell lines. a By RT-PCR SPAG6 mRNA was found to be downregulated in NSCLC cell lines but not in NHBECs. In contrast, SPAG6 methylation was observed in all NSCLC cell lines with downregulated mRNA expression but not in NHBECs by MS-HRM analysis. b SPAG6 methylated cells were treated with 5-AzadC alone or with a combination of 5-AzadC and TSA. RT-PCR analyses revealed upregulation of SPAG6 expression after drug treatment. Fold changes of gene expression in treated cells compared to untreated cells are shown. c Results from BGS of a part of the SPAG6 5′ region in NHBECs, A549 and NCI-H1975 cells are shown. 56 CpG sites (indicated as pink bars) were analysed for SPAG6 methylation. TSS, transcription start site, black squares indicate methylated CpG sites, white squares unmethylated CpG sites. Analyses of BGS results of the SPAG6 5′ region showed statistically significant differences of methylated CpG sites between NHBCEs and NSCLC cells (****p < 0.0001). d L1TD1 mRNA was found to be downregulated in NSCLC cell lines but not in NHBECs by RT-PCR. By MS-HRM analysis L1TD1 methylation was detected in all NSCLC cell lines with downregulated mRNA expression but not in NHBECs. e L1TD1 methylated cells were treated with epigenetically active drugs. RT-PCR analyses revealed upregulation of L1TD1 expression after drug treatment. Fold changes of gene expression in treated cells compared to untreated cells are shown. f Results from BGS of a part of the L1TD1 5′ region in NHBECs, A549 and NCI-H1650 cells are shown. Twenty-four CpG sites were analysed for L1TD1 methylation. TSS, transcription start site, black squares indicate methylated CpG sites, white squares unmethylated CpG sites and grey squares intermediate methylation. Analysis of BGS results of the L1TD1 5′ region showed statistically significant differences of methylated CpG sites between NHBCEs and NSCLC cells (****p < 0.0001)