Fig 1.
Effect of zebularine on cell viability in CCA cell lines.
TFK-1 (A), HuCCT1 (B), KKU-100 (C), KKU-M156 (D) and KKU-M213 (E) cells were treated with zebularine at indicated concentrations for 72 h. Cell growth was measured by WST assay. Data are the means ± SEM of results from at least three independent experiments. *p < 0.05, compared to 0 μM.
Fig 2.
Effect of zebularine on DNMT expression in CCA cell lines.
The protein levels of DNMT1, DNMT3a, and DNMT3b in TFK-1 (A) and HuCCT1 (B) cells after zebularine treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the protein levels of DNMT1, DNMT3a, and DNMT3b. GAPDH was used as a loading control.
Fig 3.
Effect of DNMT1 knockdown on cell viability in CCA cell lines.
The forward transfection of control siRNA (control) as the negative control, siDNMT1#1 or siDNMT1#2 at indicated concentrations was performed; after 72 h of transfection, the protein levels of DNMT1, DNMT3a, DNMT3b and GAPDH were detected by western blot analysis, and cell growth was measured by WST assay in TFK-1 (A) and HuCCT1 (B). Data are the means ± SEM of results from at least three independent experiments. *p < 0.05, compared to control.
Fig 4.
Effects of zebularine on apoptotic cell death in TFK-1 cells.
TFK-1 cells were treated with zebularine at indicated concentrations for 72 h. Apoptosis was measured using apoptotic/necrotic/healthy cell detection assay (A) and Cell Death Detection ELISA assay (B). The data are expressed as fold-increases relative to the respective untreated samples. Caspase-3/7 (C), -8 (D), and -9 (E) activities were determined using Caspase-Glo Assays. The data are expressed as fold-increases relative to the respective untreated samples (RLU/60 min/mg protein). (F) Forward transfection of negative control siRNA (NC) as the control, siDNMT1#1 or siDNMT1#2 at 30 nM was performed, and apoptosis was measured by Cell Death Detection ELISA assay. Data are the means ± SEM of results from at least three independent experiments. *p < 0.05, compared to 0 μM.
Fig 5.
Effects of zebularine on apoptotic cell death in HuCCT1 cells.
HuCCT1 cells were treated with zebularine at indicated concentrations for 72 h. Necrosis (A) and apoptosis (B) were measured using apoptotic/necrotic/healthy cell detection assay. (C) Apoptosis also was measured by Cell Death Detection ELISA assay (C). The data are expressed as fold-increases relative to the respective untreated samples. Caspase-3/7 (D), -8 (E), and -9 (F) activities were determined using Caspase-Glo Assays. The data are expressed as fold-increases relative to the respective untreated samples (RLU/60 min/mg protein). (G) Forward transfection of negative control siRNA (NC) as the control, siDNMT1#1 or siDNMT1#2 at 30 nM was performed, and apoptosis was measured by Cell Death Detection ELISA assay. Data are the means ± SEM of results from at least three independent experiments. *p < 0.05, compared to 0 μM.
Fig 6.
Effects of zebularine on cell growth in CCA cell lines.
TFK-1 and HuCCT1 cells were treated with zebularine at indicated concentrations for 24 h. Cell growth was measured by WST assay in TFK-1 (A) and HuCCT1 (B) cells. Apoptosis was measured by Cell Death Detection ELISA assay in TFK-1 (C) and HuCCT1 (D) cells. Uptake of BrdU was measured by ELISA in TFK-1 (E) and HuCCT1 (F) cells. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM.
Fig 7.
Effect of zebularine on DNA methylation and β-catenin expression in CCA cell lines.
Scatter plot of the average beta values at 482,143 CpG sites for zebularine-treated (y-axis) and control (x-axis) TFK-1 (A) and HuCCT1 (B) cells (n = 3 for each group). Dots for CpG sites whose delta-beta value is >0.1 or <-0.1 are shown in green (59 [0.012%] hypermethylated and 151,482 [31.4%] hypomethylated CpG sites in TFK-1 cells and 70 [0.015%] hypermethylated and 47,438 [9.8%] hypomethylated CpG sites in HuCCT1 cells). The protein levels of β-catenin in TFK-1 (C) and HuCCT1 (D) cells after zebularine treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the protein levels of β-catenin. GAPDH was used as a loading control.
Fig 8.
Effect of zebularine on DNA damage in CCA cell lines.
The DNA damage in TFK-1 (A) and HuCCT1 (B) cells after zebularine treatment for 72 h at different concentrations. After treatment, DNA samples were extracted and DNA damage assay was performed. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM.
Fig 9.
DNMT1 is upregulated in CCA tissues.
DNMT1 immunoreactivities in human normal liver (A, C) and CCA tissues (B, D). A and B are derived from a single patient (patient #1; male, 74 years old), and C and D are derived from another single patient (patient #2; female, 67 years old). The immunohistochemical analyses were performed for DNMT1 protein in paraffin-embedded sections. Bar; 100 μm.