Fig. 3.

The butyrate-induced Wnt activity is augmented by Dll1icd in HCT-116 CC cells. (A) Representative Western blot analyses of HCT-116 CC cells transiently transfected with pcDNA3.1 or a construct expressing Dll1icd. The exogenous protein was detected with a Dll1-specific antibody (sc-8155, Santa Cruz Biotechnology). (B) Dll1icd augments the induction of Wnt signaling by butyrate, as measured with the TopFlash and FopFlash reporter constructs. HCT-116 cells were co-transfected transiently with one of the Wnt signaling reporters (TopFlash or FopFlash), pcDNA3.1 or Dll1icd-expression vector in a 96-well plate. pRL-TK was co-transfected as a measure for transfection efficiency. The ratio between the reporters and the effector vectors was 1:3, the total amount DNA per well was 0.32 μg. Transfections were carried out with Lipofectamine 2000, and treatment with 5 mM butyrate was for 17h. Each transfection was performed in duplicate wells; data represent the mean from results of at least three experiments. Wnt signaling was calculated as the ratio TopFlash/FopFlash (Top/Fop). There was no statistically significant difference between Wnt activity in mock-treated cells transfected with pcDNA3.1 or Dll1icd-expressing construct. For butyrate-treated cells, Top/Fop was 35.3±6.6 and 52.4±6.9 (P<0.05) for pcDNA- and Dll1icd-transfected cells, respectively. (C) Dll1icd augments the induction of Wnt signaling by butyrate, as measured with the Lef-OT and Lef-OF reporter constructs. The transfections were performed as described in 3B. The ratio Lef-OT/Lef-OF is a measure of Wnt activity. There was no significant difference between Wnt levels in mock-treated cells transfected with empty vector or Dll1icd-expressing construct. In butyrate-treated cells, Lef-OT/Lef-OF was 16.3±2.7 and 34.8±2.4 (P<0.05) for pcDNA- and Dll1icd-transfected cells, respectively.