Figure 8.

Knockdown of DDX3 suppresses p53 expression by inhibiting p53 mRNA translation but not p53 stability. (a) Western blot analysis reveals DDX3 knockdown repressed p53 expression in HCT116, 293 T and U2OS cells. Original images of western blots were presented in Supplementary Fig. S6a. (b) Western blot analysis reveals dose-dependent knockdown of DDX3 resulted in differential repression of p53 or p21 in HCT116 and 293 T cells. Original images of western blots were presented in Supplementary Fig. S6b. (c) Western blot analysis reveals ectopic expression of shDDX3-resistant Flag-DDX3 partially rescued p53 or p21 in the DDX3-knockdown HCT116 and 293 T cells. Original images of western blots were presented in Supplementary Fig. S6c. (d) Western blot analysis reveals DDX3 depletion resulted in reduced p53 degradation. Control and DDX3-knockdown HCT116 cells were incubated with 40 μg/ml cycloheximide for indicated time points and the half-life of p53 was analyzed. Original images of western blots were presented in Supplementary Fig. S7a. (e) Schematic diagrams of luciferase reporter RNAs. T7-pA and 5′ + 3′-pA are uncapped reporter RNAs whereas Cap-T7-pA and Cap-5′ + 3′-pA are capped reporter RNAs. In the 5′ + 3′-pA and Cap-5′ + 3′-pA reporter RNAs, the p53 IRES (134 bp) is inserted upstream and p53 3′UTR (1.2k bp) is inserted downstream of the firefly luciferase gene (FL). Knockdown of DDX3 suppressed the p53 IRES-mediated translation and cap-dependent translation of reporter RNAs. Data are shown as average value ± S.D. calculated from three independent experiments performed in triplicate. **P < 0.01; *P < 0.05.