Figure 8: CD24 silencing restores transcriptional activity of p53.

(a) Effect of CD24 silencing on expression of NPM, ARF, MDM2 and p53. Scrambled (Scr) or CD24-shRNA-silenced DU145 cells were analysed for CD24, NPM, ARF, MDM2 and p53 by western blot. (b) In ARF-deficient U2OS cells, ectopic expression of CD24 had no effect on the levels of MDM2 and p53. Actin levels were used as loading control. (c) Endogenous mutant p53 is responsible for the upregulation of p21 in CD24-silenced DU145 cells. (d) In DU145 cells, silencing CD24 restores transcriptional induction of the p21 promoter. DU145 cells transduced with Scr or shRNA for eitherTP53 or CD24 were transfected with a luciferase reporter containing the p21 promoter. The WT reporter has an intact p53-binding site, whereas the mutant promoter has the site deleted. (e,f) CD24 silencing restores transcriptional activity of multiple TP53 mutants. DU145 cells in which the endogenous TP53 was silenced by shRNA were transduced with Scr or CD24 shRNA lentivirus. The stable cell lines were then transfected with p53 mutants in conjunction with WT or mutant p21 promoter reporter constructs. p53 levels (e) were determined by western blot, while luciferase activity (f) was measured at 72 h after transfection. The mutants used were p53^R273H, p53^V143A, p53^R280T and p53^R175H. Numbers on the x axis indicate the amino-acid positions of the p53 mutations. (g) As in e and f, except that the endogenous p21 and MDM2 mRNA were measured by quantitative PCR. Data shown are means and s.d. of triplicate samples. All data have been reproduced two or three times.