Figure 2

β-catenin/TCF activation by DDX3 is mediated through inhibition of β-catenin degradation by PP2A.

(a) CCM2 and T84 cells were transfected with two kinds of shDDX3 and two doses of DDX3-overexpression vector. These cells were incubated with 5 μmol/L MG132 for an additional 5 h and then the cell lysates were analyzed by immunoblotting with anti-β-catenin antibody. (b) The CCM2 cells were transfected with the indicated combination of TCF promoter plasmid, shDDX3, shCK1ε and shDvl2 for 48 h. The T84 cells were transfected with the indicated combination of the TCF promoter plasmid, the DDX3-overexpression plasmid, shCK1ε and shDvl2 for 48 h. The expression of DDX3, CK1ε, Dvl2, pDvl2, β-catenin, pβ-catenin (Ser33, Ser37 and Thr41), GSK-3β, pGSK-3β (Ser9), PP2A (A subunit) and β-actin were determined by western blotting using their specific antibodies. The TCF promoter activity was evaluated by a luciferase reporter activity assay. (c) The CCM2 cells were transfected with shDDX3, shCK1ε, or shDvl2 for 48 h. The T84 cells were transfected with the indicated combination of DDX3-overexpression plasmid, shCK1ε and shDvl2 for 48 h. These cells were then treated with MG132 for 5 h. The cell lysates were immunoprecipitated with anti-β-catenin–conjugated beads to analyze by immunoblotting with anti-PP2A and anti-β-catenin antibody. (d) The CCM2 cells were transfected with shDDX3, shCK1ε, or shDvl2 for 24 h. The T84 cells were transfected with the indicated combination of DDX3-overexpression plasmid, shCK1ε and shDvl2 for 24 h. The invasion capability of the indicated cells was determined by Boyden chamber assays. The invasion ability and the TCF promoter activity of these cells with different treatments are shown as fold changes compared with their NC and VC. All experiments were performed three independent times. The mean values and the standard deviations are indicated as columns with error bars. The P value was statistically determined by the Student’s t-test. *P < 0.05 compared with vector (VC) or non-specific shRNA controls (NC). #P < 0.05 compared with DDX3-overexpressing T84 cells. The samples were derived from the same experiment and gels/blots were processed in parallel. Full-length blots are presented in Supplementary information Figure S1 and S2.