Figure 2. Zhangfei regulates p53 in a post-translational level and induces p53 nuclear localization. (A) Zhangfei enhances the expression of p21, a p53-dependant gene. D-17 cells were mock-infected or infected with either Adeno-ZF or Adeno-LacZ, 24 h after infection, transcripts for p53, p21, PIG3, and NOTCH1 were determined by qRT-PCR. The values represented fold changes of transcripts between Adeno-LacZ (white bar) or Adeno-ZF (black bar) infected cells with mock-infected cells. Error bars indicated standard deviations from means. (B) Zhangfei stabilizes p53 and p21 proteins. p53, p21, Zhangfei, and GAPDH were detected by immunoblotting in D-17 cells either mock-infected (MI) or infected with either Adeno-LacZ or Adeno-ZF. As a positive control, cells were treated with 0.5 µM doxorubicin for 6 h. (C) Zhangfei activates p53-dependent transactivation. D-17 cells were transfected with a reporter plasmid containing the coding sequence for CAT linked to a promoter with 2 copies of p53-responsive element (pCAT3B-p53RE, 0.5 µg) in the presence or absence of a plasmid expressing Zhangfei (pcZF, 1 µg). The promoter-less parental reporter plasmid, pCAT3B was included as a control to show basal CAT activity. All samples also contained, as a control, a plasmid expressing β-galactosidase. 24 h after transfection, the CAT activity was determined. Values represented the relative CAT activity (normalized to the internal control, β-galactosidase) of different treatments. Standard deviations from means of three individual experiments are and the significant P values from ANOVA tests were noted above the bars. (D) Zhangfei alters the subcellular localization of p53. D-17 cells were transfected with 1 µg of pcZF or a control (pcDNA3), and 12 h and 36 h after transfection, endogenous p53 as well as Zhangfei were visualized by immunofluorescence with anti-p53 and anti-ZF antibody. The nucleus was detected by Hoechst staining (bar = 10 µm). The means and standard deviations of representative experiments (n = 3) were shown. P < 0.05 were considered to be significant.