Figure 1.
The effect of zebularine on HepG2 cell viability.
HepG2 cells were treated with zebularine at indicated concentrations for 72 h (A) and 24 h (B). Cell growth was measured by WST assay. (C) HepG2 cells were treated with zebularine at indicated concentrations for 24 h. Uptake of BrdU was measured by ELISA. (D) HepG2 cells were treated with zebularine at indicated concentrations for 72 h. Apoptosis was measured by TUNEL assay. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM.
Figure 2.
Effect of zebularine on the DNMTs expression and DNA methylation in HepG2 cells.
(A) The protein level of DNMT1, DNMT3a, and DNMT3b 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 level of DNMT1, DNMT3a, and DNMT3b. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM. (B) Scatter plot of the average beta values at 485,415 CpG sites for zebularine-treated (y-axis) and control (x-axis) HepG2 cells (n = 3 for each group). Dots for CpG sites whose delta-beta value is >0.1 or <−0.1 are shown in green (35 [0.0072%] hypermethylated and 162 [0.033%] hypomethylated CpG sites).
Figure 3.
Effects of zebularine on the protein expression of cell-cycle regulator.
The protein level of CDK2, p-Rb, and Rb after zebularine treatment for 24 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the protein level of CDK2, p-Rb, and Rb. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM.
Figure 4.
Effects of zebularine on the protein expression of p21WAF/CIP1 and p53.
The expression of p21WAF/CIP1 and p53 after zebularine treatment for 24 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the protein level of p21WAF/CIP1 and p53. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM.
Figure 5.
Effects of zebularine on phosphorylation of p44/42 MAPK.
The phosphorylation and expression of p44/42 MAPK after zebularine treatment for 24 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the phosphorylated and total p44/42 MAPK protein level. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM.
Figure 6.
The effect of zebularine on apoptosis-related proteins.
HepG2 cells were treated with zebularine at indicated concentrations for 72 h. (A) Caspase-3/7, -8, and -9 activities were determined using Caspase-Glo Assays. The data are expressed as fold-increase relative to the respective untreated samples (RLU/60 min/μg protein). (B) The protein level of Bax and Bcl-2 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 level of Bax and Bcl-2. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM.
Figure 7.
Effects of zebularine on phosphorylation of PKR.
(A) The phosphorylation and expression of PKR after zebularine treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the phosphorylated and total PKR protein level. GAPDH was used as a loading control. *p<0.05, compared to 0 μM. (B) Effect of the overexpression of PKR in zebularine-induced cell death. The forward transfection of the empty vector (Halo Tag control vector) as the control or the plasmid-containing PKR cDNA sequence (pFN21A-hPKR) was performed, and the cells were then treated with different concentrations of zebularine for 72 h. *p<0.05, compared to control. Data are the means ± SEM of results from at least three independent experiments.
Figure 8.
Effects of zebularine on phosphorylation of PKR in HeLa cells.
(A) HeLa cells were treated with zebularine at indicated concentrations for 72 h. Cell growth was measured by WST assay. (B) The phosphorylation and expression of PKR after zebularine treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the phosphorylated and total PKR protein level. GAPDH was used as a loading control. *p<0.05, compared to 0 μM.