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. 2016 Mar 17;7(16):22687–22699. doi: 10.18632/oncotarget.8143

Figure 3. DDX3-mediated β-catenin activation is responsible for cell invasion via increasing ZEB1 expression.

Figure 3

(A) The TCF promoter activity (TOP flash, WT and FOP flash, Mut) in DDX3-knockdown in CCM2 cells and DDX3-overexpressing T84 cells was evaluated by luciferase reporter assay. (B) CCM2 and T84 cells were transfected with two kinds of DDX3 shRNA and two doses of DDX3 expression vector. These cells were incubated with 5 μM of MG132 for an additional 5 hr, and then the cell lysates were analyzed by SDS-PAGE, followed by immunoblotting with anti-β-catenin antibody. (C) CCM2 cells were co-transfected with the indicated combinations of shDDX3, shKRAS and mutant KRAS (G12V) for 24 hr. T84 cells were co-transfected with the indicated combinations of DDX3 and shKRAS Consequent, these cells were treated with or without 10 μM LY294002. After treatment for 4 hr, the cells lysates were separated by SDS-PAGE for the protein expression using western blotting. (D) CCM2 and T84 cells were transfected with DDX3 shRNA and its expression vector for 48 hr. These cells were separated with cytosolic and nuclear fraction to evaluate β-catenin protein expression by SDS-PAGE using a western blotting. (E) CCM2 cells were transfected with shDDX3 or shβ-catenin for 24 hr. T84 cells were transfected with DDX3 expression vector or combined with shβ-catenin transfection for 24 hr. The expression of DDX3 and β-catenin in these treated both cells were evaluated by western blotting and the invasion capability was determined by Boyden chamber assay. The efficacy of invasion ability was compared with NC and VC, respectively. (F) Two kinds of shDDX3 were transfected into high DDX3 expressing CCM2 cells and two doses of DDX3 expression vector were transfected into low DDX3 expressing T84 cells. After 48 hr, the cell lysates were used for evaluating the expression of DDX3, ZEB1, Snail, Slug, and TWIST by Western blotting. ZEB1 mRNA expression was evaluated by real-time PCR. (G) CCM2 cells were transfected with shDDX3, shβ-catenin, shZEB1 and β-catenin inhibitor (XAV939) singly or in combination, for 24 hr. T84 cells were transfected with DDX3 expression vector, β-catenin, ZEB1-knockdown plasmids, and β-catenin inhibitor (XAV939) singly or in combination, for 24 hr. The invasion capability in both cells with different treatments were evaluated by Boyden chamber assay for 16 hr. (H) Example of lungs of mice with visual lung tumor nodules at 6 weeks after tail vein inoculation of indicated cells. Representative Hematoxylin and Eosin staining was presented in lung tumor nodules from each group of mice. The number of lung tumor nodules of each group of mice. Data are presented as mean ± SD. ZEB1 expressions in lung tumors were evaluated by IHC using a specific antibody.