## 2 Results
### Epigenetic Regulation of SLC10A1/NTCP  
Because genetic variants seem to have only a minor impact on _SLC10A1_/NTCP expression and function, we next evaluated the impact of DNA methylation. First, functional evaluation of the epigenetic regulation of _SLC10A1_/NTCP expression was performed using cell culture experiments. HepG2 and HuH-7 cells were treated with AZA (5-Aza-2\({}^{\prime}\)-deoxycytidine, decitabine), which is a well-established DNA methylation inhibitor. Subsequently, mRNA expression of _SLC10A1_ was analyzed using real-time PCR technology. As shown in Figure 5A, treatment of both HepG2 and HuH-7 cells with 1 uM AZA led to an increase of _SLC10A1_ mRNA expression. Calculated as a multiple of the DMSO control, the AZA treatment led to a 5.4-fold elevation of the _SLC10A1_ transcript level in HepG2 cells and a 16.3-fold increase in HuH-7 cells. In addition, global DNA methylation status was determined using an established liquid chromatography with tandem mass spectrometry (LC-MS/MS) method [62] in order to verify the demethylating effect of AZA treatment. As indicated in Figure 5B, the proportion of 5-methylcytosine residues in genomic DNA of HepG2 and HuH-7 cells decreased after treatment. To verify the influence of DNA methylation on _SLC10A1_ promoter activity, different promoter/reporter fusion plasmids containing either methylated or mock-methylated _SLC10A1_ promoter fragments were investigated (Figure 5C). Reporter gene constructs, in which the firefly luciferase gene was under the control of \(\sim\)1 kb or \(\sim\)2 kb _SLC10A1_ promoter fragments, were transfected into HuH-7 cells. The obtained luciferase intensities of the lysates originating from cells transfected with unmethylated promoter-reporter gene constructs were set as 100%. As shown in Figure 5C, DNA methylation resulted in a reduction of luminescence of 44.8% for the methylated \(\sim\)2 kb promoter fragment. For the methylated \(\sim\)1 kb promoter fragment, the luminescence was reduced by 64.7%. Thus, reporter constructs with a methylated _SLC10A1_ promoter fragment showed significantly reduced promoter activity compared to constructs with an unmethylated promoter, confirming the influence of DNA methylation on _SLC10A1_ promoter activity. Based on the results of the cell culture experiments, _SLC10A1_ DNA methylation levels of individual CpG sites in the promoter and exon 1 region, as well as in a predicted CpG island of intron 1, were investigated using MALDI-TOF MS. As shown in Figure 5D, the DNA methylation was highly variable in the _SLC10A1_ promoter and exon 1 gene region, whereas the CpG island in intron 1 was hypermethylated in human liver. Correlation analyses of DNA methylation levels and mRNA or protein levels (Table 2) indicated a significant association between DNA methylation at specific CpG sites and the protein levels even after Benjamini-Hochberg correction for multiple testing (5_CpG_5, adjusted \(p\) = 0.03; 5_CpG_2, adjusted \(p\) = 0.03). Figure 5: Epigenetic regulation of _SLC10A1_/NTCP. (**A**) Effect of 5-Aza-2’-deoxycytidine (AZA) treatment on _SLC10A1_ mRNA expression. Cells were cultured with 1 \(\mu\)M AZA and mRNA levels (normalized to _ß-actin_) were determined using TaqMan technology. Fold increase in expression compared to untreated cells was calculated. (**B**) Effect of treatment with 5-Aza-2’-deoxycytidine (AZA)  
on global DNA methylation. Cells were either untreated or treated with 1 uM AZA and the amount of 5-methylcytosine was quantified using LC-MS/MS to verify the effect of AZA treatment on global DNA methylation. Results represent mean of at least 2 experiments \(\pm\) SE. (**C**) Scheme of the _SLC10A1_ gene locus showing the examined promoter region. Two promoter fragments (short and long fragment), were cloned and reporter activity depending on methylation status of the promoter fragments was investigated in HuH-7 cells. Relative activities of the methylated fragments are shown compared to the mock-methylated fragments, whose activities were set to 100%.