Fig 2: Kaplan–Meier analysis of the relationships of intratumoral expression of biomarkers with progression-free survival in patients with biliary tract cancers. (A) hENT1, (B) DCK, (C) CDA, (D) RRM1. hENT1, human equilibrative nucleoside transporter 1; DCK, deoxycytidine kinase; CDA, Cytidine deaminase; RRM1, ribonucleotide reductase subunit 1.

Fig 3: The expression levels of CDA, DCK, UCK1 and UCK2 mRNA and the methylation status at the 5'-flanking regions of the CDA gene before and after 5AC treatment in three matched clinical samples(A–D) The mRNA expression levels of CDA (A), DCK (B), UCK1 (C) and UCK2 (D) in tumor samples of patients with MDS before (Pre-5AC) and after (Post-5AC) treatment were determined by a quantitative RT-PCR. The expression level in Pre-5AC samples of case 2 was set to 1 (E) The methylation status of the 5'-flanking region of the CDA gene in clinical samples from three patients with MDS before (Pre-5AC) and after (Post-5AC) treatment (cases 2, 4 and 6). The (UM) and (M) bands represent the unmethylated and methylated bands, respectively. The CpG sites that were analyzed are shown in Figure 3C.

Fig 4: Treatment with 5AC induced the expression of CDA, together with the demethylation of the CpG site in the 5′-flanking region of the CDA gene(A) The mRNA expression levels of CDA, DCK, UCK1 and UCK2 in MDS-L cells after treatment with various doses of 5AC for 48 h were determined by a quantitative RT-PCR. Each mRNA level was normalized to the rRNA level, and the value relative to that in untreated cells was shown. The expression level in untreated cells was set to 1. (B) The mRNA expression levels of CDA, DCK, UCK1 and UCK2 in MDS-L cells after treatment with DAC for 48 h were determined. Each mRNA level was normalized to the rRNA level, and the value relative to that in untreated cells was shown. The expression level in untreated cells was set to 1. (C) A schematic representation of the potential CpG sites in the 5′-flanking region of the CDA gene. (D) The effect of DAC or 5AC on the methylation status of the 5′-flanking region of CDA gene. Cells were treated with DAC or 5AC for 48 h. The (UM) and (M) bands represent the unmethylated and methylated bands, respectively. (E) Comparison of luciferase activity driven by CDA promoter. The cells were treated with 5AC at 100 nM for 48 h. The relative promoter activity was presented as ratio to the activity in control vector-transfected cells.

Fig 5: The IHC analysis of clinical samples from patients with MDS(A) The IHC analysis of CDA in bone marrow biopsy samples from patients with MDS before (Pre-5AC) and after (Post-5AC) treatment. (B) The quantitative analysis of the CDA expression levels in patients with MDS before and after 5AC treatment, based on the IHC analysis. (C) The IHC analysis of DCK in bone marrow biopsy samples from patients with MDS before and after 5AC treatment. (D) The quantitative analysis of the DCK expression in patients with MDS before and after 5AC treatment, based on the IHC analysis.