Figure 6. SPDEF inhibits β-catenin transcriptional activity through protein-protein interaction.
(A) Human colon cancer lines were co-transfected with TOPflash or FOPflash, with Flag-tagged SPDEF and Renilla plasmids. β-catenin activity was detected by the dual luciferase assay 48h post-transfection. Quantitated result of relative β-catenin activity derived from three independent experiments was shown. (B) HEK-293T Super TOPflash cells were transfected with different amounts of Flag-tagged SPDEF plasmids as indicated. Canonical Wnt/β-catenin pathway was activated by Wnt3a conditional medium 24h post-transfection. 48h post-transfection, the β-catenin activity was detected. Quantitated result of relative β-catenin activity derived from three independent experiments was shown. Immunoblot analysis showed the expression level of ectopic SPDEF and endogenous β-catenin. (C) HEK-293T Super TOPflash cells were transfected with the indicated plasmids. 48h post-transfection, the β-catenin activity was detected. Quantitated result of relative β-catenin activity derived from three independent experiments was shown. (D) Schematic illustration of truncated Flag-tagged human NLS-SPDEF mutants. HEK-293T Super TOPflash cells were transfected with indicated NLS-SPDEF mutants. Canonical Wnt/β-catenin pathway was activated by Wnt3a conditional medium 24h post-transfection. 48h post-transfection, the β-catenin activity was detected. (E) Human cancer colonoids stably transduced with pINDUCER20 expressing Flag-tagged human NLS-SPDEF mutants, and mock control were generated. Bright field images were taken after SPDEF induction as indicated by hours. Quantitated result indicated SPDEF WT and Δ1 inhibit cancer colonoid growth. Colonoid sizes (maximum diameter3) were normalized to the average size (maximum diameter3) of mock control after SPDEF induction at 24h. Error bars, S.E.M.. ***, p<0.001. **, p<0.005.