Fig. 2. Confirmation of Smad and LEF1 transcriptional activities. (A) p3TP-Lux reporter gene assay demonstrated that Smad transcriptional activity increased from unfused to fused MEE cells, with a sharp increase in activity upon stimulation with TGF3. A dominant-negative Smad4 (DN Smad4) construct inhibited luciferase activity (mean ± s.d.; n=3; ^*P<0.05 compared with Unfused; ^**P<0.05 compared with p3TP-Lux). (B) pTOPFLASH-Lux (TF) reporter gene assay showed that LEF1 transcriptional activity continually increases as TGF3 signaling progresses. Mutated LEF1 binding sites of the pFOPFLASH-Lux reporter, as well as treatment with DN Smad4 (Smad4 being necessary for LEF1 gene expression) or dominant-negative LEF1 (DN LEF1) inhibited transcriptional activity whereas antisense -catenin/-catenin oligodeoxynucleotide (AS B-catenin ODN) did not (mean ± s.d.; n=3; ^*P<0.001 compared with Unfused; ^**P<0.01 compared with TF).