A. Heat map showing changes in the transcript levels of genes in the Wnt signaling network. HEK 293 cells were serum-starved for 24 hours and stimulated for different lengths of time with 200 ng/mL Wnt3a. Transcript levels were measured by qPCR and normalized to the average of five housekeeping genes. Changes are relative to serum-starved cells (0 h of stimulation). B. Genes with similar dynamic expression profiles were grouped together using self-organizing maps. Most canonical Wnt response genes fell into group IV, whereas negative regulators of Wnt signaling fell into groups III and VI.

A. Correlations were calculated between time-dependent expression profiles and either proliferation (blue) or migration (red). The dotted line indicates a P-value of 0.05 (* P<0.05). B, C. Transcript levels of CtBP1 and c-Myc in human colorectal carcinomas. Left: the relative levels of CtBP1 or c-Myc mRNA in normal colon tissue and in colorectal cancers of different stages. Horizontal bars indicate the mean of each group. Right: the relative levels of CtBP1 and c-Myc mRNA were significantly higher in colon cancer tissue relative to normal mucosa (* P<0.05; Kruskal-Wallis test). Error bars indicate the standard deviation. Data for Casein kinase 1α1, c-Jun, and Brachyury are provided in Figure S6.

A. Simplified schematic of the canonical Wnt signaling network. Only proteins whose transcript levels changed by more than two-fold for at least one time point during the 24-hour time-course are shown. Cue and signal antagonists are outlined in orange; response antagonists are outlined in purple; transcriptional response genes are colored blue. B, C. Simplified schematics of the Wnt signaling network, highlighting proteins whose genes were upregulated (green) or downregulated (red) by more than two-fold after (B) 1 hour or (C) 12 hours of stimulation with 200 ng/mL Wnt3a. Schematics showing changes at all six time-points are provided in Figure S4. D. Plot of changes in the transcript levels of the proteins highlighted in panel (A). Cue and signal antagonists and shown in orange; response antagonists are shown in purple; transcriptional response genes are shown in blue. (E, F) The transcriptional response to Wnt3a stimulation is characterized by two distinct phases: an early phase (1–3 hours) and a late phase (3–24 hours). E. In the early phase, genes encoding cue and signal antagonists are downregulated, driving the upregulation of signal and response genes. F. In the late phase, genes encoding cue, signal, and response antagonists are upregulated, attenuating the expression of transcriptional response genes.

A. Western blots reveal progressively higher levels of β-catenin, c-Myc, and CtBP1 in colon cancer cell lines of increasing tumorigenicity (Caco-2<DLD-1<HT-29<HCT116<SW480), relative to the normal colon cell line, CCD-18Co. B. Quantification of the Western blot data in panel A. All values were normalized to β-actin levels. C. Roles of CtBP1 and c-Myc in regulating gene expression. HEK 293 cells were transiently transfected with control siRNA or siRNAs targeting either CtBP1 or c-Myc. After 48 hours, the cells were serum-starved for 24 hours and stimulated with 200 ng/mL Wnt3a for 12 hours. Transcript levels were measured by qPCR. Knocking down either CtBP1 or c-Myc resulted in decreased levels of β-catenin. Data are the mean of two independent experiments and error bars indicate the standard error of the mean (* P<0.05; one-way ANOVA). D. Pathway diagram illustrating the roles of CtBP1 and c-Myc in Wnt3a-mediated activation of canonical Wnt signaling. The levels of both CtBP1 and c-Myc are upregulated in response to Wnt3a stimulation. In the absence of Wnt3a, CtBP1 acts a transcriptional repressor and inhibits the expression of MYC. In the presence of Wnt3a, both CtBP1 and c-Myc drive Wnt signaling by upregulating β-catenin. E. Distribution of CtBP1 immunoreactivity scores in normal mucosa and in colorectal adenocarcinomas determined using tissue microarrays (n = 75 cases; 150 tumor cores). The immunoreactivity of CtBP1 was classified using a four-grade scale: 0, fewer than 10% positively stained cells; 1, between 10% and 25%; 2, between 25% and 50%; and 3, greater than 50%. F. Representative images from the tissue microarrays, showing patient-matched normal and cancerous tissue from stage II and stage IV colorectal carcinomas (10× magnification).