Figure 4

G244S mutation disrupted the p53 function.

(A) G244S mutation increased the half-life of p53. TP53-deficient HCT116 cells were transiently transfected with plasmids expressing either the WT or G244S p53. 48 h later, cells were treated with cycloheximide (CHX) for 0, 1, 2, 4, 6 and 8 h. Equal amounts of whole cell lysates were analyzed by Western blot with a p53 antibody. Actin was used as an internal control. (B) The graph illustrates the quantification of WT and G244S p53 by densitometry of triplicate experiments (mean ± SEM). *p < 0.05, **p < 0.01 by post hoc Bonferroni t test. (C) The subcellular localization of WT and G244S p53 proteins as analyzed with immunofluorescence. Scale bar = 10 μm. (D) Transcriptional activities of WT and G244S p53 as determined by luciferase activity assays. The luciferase reporter plasmid p21-Luc were transiently transfected into TP53-deficient HCT116 cells in combination with WT or G244S p53 expressing plasmids. **p < 0.01 by post hoc Dunnett’s t test (E) The dominant negative effect of G244S p53 on WT p53. Increasing amount of G244S p53-expressing plasmid was cotransfected with a fixed amount of WT p53-expressing plasmid and the p21-Luc into TP53-deficient HCT116 cells. The basal level of luciferase was set as 1. Data from all other transfection are presented as fold induction above this level. Each value was the mean ± SEM of three replicates from a single assay. The results shown were representative of three independent experiments. **p < 0.01 by post hoc Bonferroni t test. (F) The induction of p21 after transfection of WT or G244S p53-expressing plasmids in TP53-deficient HCT116 cells. The protein levels of p53, p21 and β-actin was analyzed with immunoblotting. (G) The ability of WT and G244S p53 to bind corresponding DNA as assayed with a chromatin immunoprecipitation (ChIP) assay in TP53-deficient HCT116 cells. (H) The growth curve of SaoS2 cells stably transfected with WT or G244S TP53, as measured with a MTT assay. The cell numbers at day 1 was set to 1. **p < 0.01, post hoc Bonferroni t test.