Fig 2: A Zbed3 mutant lacking an Axin binding site does not increase ß-catenin expression in NCI-H1299 and A549 lung cancer cells. Overexpression of wild-type Zbed3 increased ß-catenin and p120ctn-1 expression. Transfection of the Zbed3 mutant failed to increase ß-catenin expression. The ability of regulating p120ctn-1 was also significantly inhibited when transfected with the Zbed3 mutant compared to the wild-type Zbed3. The experiments were repeated three times (*P < 0.05)

Fig 3: Overexpression of Zbed3 increases A549 and NCI-H1299 lung cancer cell proliferation and invasiveness. MTT assays show that Zbed3 overexpression promotes cell proliferation (A). Transwell assays show that Zbed3 overexpression enhanced the invasiveness of the cells (B). Dual-luciferase assays show that Zbed3 overexpression increases activity in the canonical Wnt signalling pathway (C). The experiments were repeated three times (*P < 0.05)

Fig 4: Overexpression of Zbed3 increases levels of Wnt signalling molecules in lung cancer cells. Western blots show Wnt signalling molecules, including Axin, GSK-3ß, and ß-catenin, are expressed in both A549 and NCI-H1299 cells (A). Zbed3 expression is up-regulated significantly in A549 cells 48 h after transfection (B). Zbed3 overexpression up-regulates expression of ß-catenin and its downstream mediators, including cyclin-D1 and MMP7, in A549 and NCI-H1299 cells (C). The experiments were repeated three times (*P < 0.05)

Fig 5: Treatment with a GSK3ß inhibitor abolished Zbed3's ability to increase ß-catenin expression and Wnt signalling activity. Western blots show that Zbed3 overexpression increases levels of ß-catenin while decreasing levels of phospho-ß-catenin in A549 cells, and that this effect is abolished by treating the cells with the GSK3ß inhibitor TWS119 (A). Dual-luciferase assays show that Zbed3 overexpression increases activity in the canonical Wnt signalling pathway in A549 cells, but this effect too was abolished by treatment with TWS119 (B). The experiments were repeated three times (*P < 0.05)