Figure 4

β-Catenin signaling activity is attenuated in SW480 cells stably expressing Cby. (A) CbyWT, but not CbyS20A, represses TopFlash activation. SW480 stable cells were transfected in 24-well plates with 25 ng of TopFlash or mutant FopFlash reporter as well as a Renilla luciferase plasmid to normalize transfection efficiency. Luciferase activities were measured 48 h post-transfection, normalized and corrected for background by subtraction of FopFlash values from corresponding TopFlash values. Each transfection was performed in triplicate and repeated at least three times. Shown are the means ± SD from one representative experiment. Student’s t-test; *P < 0.05 when compared with vector-control cells. (B) CbyWT, but not CbyS20A, inhibits cyclin D1 expression. Whole-cell lysates from the indicated SW480 stable cells were subjected to western blot analysis for cyclin D1 and beta-actin as a loading control. (C) Quantification of cyclin D1 expression levels. The intensity of cyclin D1 bands from western blots was quantified using the NIH Image software and normalized to that of beta-actin bands. Data are the mean band intensities ± SD from at least three independent experiments. Student’s t-test; *P < 0.05 when compared with vector-control cells.