Figure 5.

Dominant negative TCF4 (DNTCF4) promotes cell survival in SW620 Spheroids treated with 5FU through decreasing DNA damage. (A) β-catenin/TCF-mediated transcriptional activity decreased as a result of DNTCF4 expression in SW620 cells transfected with plasmid pPGS dnTcf-4(deltaN41). The normalized activity of the TCF reporter (TOP-Luc) is shown. Renilla luciferase levels were used as transfection control. (B) SW620 spheroids that express dnTCF4 were treated with 5FU for 72 h. Cell viability was measured using the CellTiter Glo-3D Kit and compared to spheroids derived from cells transfected with an empty vector. (C,D) Immunoblot analysis of cyclin D1 (C), phospho-CDK2 (Th160) (C), phosphor-histone γH2AX (Ser 139) as DNA damage marker, and survivin (D) in SW620 spheroids^dnTCF4 and control spheroids treated with or without 5FU for 72 h. Ponceau Red staining for total protein was used as loading control. (E) Comparative cell cycle analysis of SW620 spheroids^dnTCF4 and control spheroids treated with or without 5FU for 72 h. Data are represented by the mean ± SEM of at least two independent tests. One-way ANOVA followed by Bonferroni’s multiple comparisons. * p < 0.05; ** p < 0.001; *** p < 0.0001.