Fig 2: ß-catenin-TCF signaling regulates GUCA2A nuclear transcription. (A, B) Luciferase constructs driven by a constitutive (SV40) promoter and containing the guanylin 3' UTR or no 3' UTR, were expressed in (A) DLD1(DNTCF) or (B) LS174T(DNTCF) cells. Luciferase was quantified with (+) or without (–) 1 µg/mL DOX for 24 hours in DLD1 cells or 48 hours in LS174T cells. (C, D) Expression constructs containing the entire human GUCA2A gene from 5' to 3' UTR, or the entire murine Guca2a gene under the control of a constitutive (ROSA) promoter were expressed in (C) DLD1(DNTCF) or (D) LS174T(DNTCF) cells. mRNA was quantified with (+) or without (–) 1 µg/mL DOX for 24 hours in DLD1 cells, or 48 hours in LS174T cells. (E) DLD1(DNTCF) cells were treated with 1 µg/mL DOX for 24 hours and (F) HT29(APC) cells were treated with 300 µM zinc for 24 hours. GUCA2A mRNA was quantified in whole cell (W), cytoplasmic (C), or nuclear (N) fractions, with fractionation confirmed by GAPDH (cytoplasmic) or histone H3 (nuclear) protein. (G, H) GUCA2A preRNA was quantified in the (G) DLD1 and (H) HT29 whole cell fractions. (A-H) Data points represent the average of 3 wells of cells from a single experiment, with the mean of 2–4 independent experiments indicated. Significance was determined by (A, B, E, F) 2-way analysis of variance or (C, D, G, H) Student’s t test with matched analysis for independent experiments on log2-transformed results. No significance was identified between any group in individual or combined experiments in A–D. Data are presented relative to noninduced cells. *P < .05; **P < .01; ***P < .001.

Fig 3: Effects of WT APC overexpression on the HER2 signaling pathway in COLO-320 cells (with mutant APC). (A) Confirmation of APC overexpression in cells transfected with pCMV_APC vector using Western blotting. (B) Activity of the Wnt/ß-catenin pathway in COLO-320 cells transfected with pCMV_APC measured using the TCF/LEF luciferase reporter assay. The reporter vectors were cotransfected into COLO-320 cells along with the pCMV empty vector (mock) or pCMV-APC. The data were standardized to the mean of the activity of mock cells. **P < 0.01 vs mock (n = 3). (C) Measurement of activated RAS using the G-LISA assay performed 48 h after the transfection with pCMV. The data were standardized to the mean of the activity of cells transfected with mock vectors. *P < 0 .05 vs mock (n = 3) (D) Western blot results showing the effects of APC overexpression on the HER2 signaling pathway in COLO-320 cells. The cells were cotransfected with pcDNA_HER2-WT/G776S vectors and/or pCMV_APC vectors. Western blotting was performed 48 h after transfection. ß-Actin served as a loading control. (E) Measurement of activated RAS using the G-LISA assay performed 48 h after transfection. The data were standardized to the mean of the activity of cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, **P < 0.01 (n = 3). (F) Activity of the Wnt/ß-catenin pathway in COLO-320 cells measured 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate. (G) Measurement of activated RAS using the G-LISA assay in COLO-320 cells 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate.

Fig 4: Suppression of cell differentiation in Apc-KO organoids. Organoids formed in the presence of WRN-CM were cultured in the presence or absence of WRN-CM for 3 more days. (A, B) The organoids were stained with anti-Mucin-2 antibody (A, green) and anti-chromogranin A antibody (B, green). Counter stain, phalloidin for F-actin (red) and DAPI for DNA (blue). The ratio of organoids containing at least one Mucin-2-positive (A) and chromogranin A-positive (B) cells to total organoids was quantified for each group. The total number of organoids analyzed is shown above each bar. Dunn’s multiple comparison test was used after Kruskal–Wallis test to calculate P values. (C) Alkaline phosphatase activity (stained purple) was detected in the organoids. The ratio of organoids containing at least one alkaline phosphatase-positive cell to total organoids was quantified for each group. The total number of organoids analyzed is shown above each bar. ND not detected. Scale bar, 100 µm.

Fig 5: Effects of APC KO on HER signaling in HeLa cells (with WT APC). (A) Confirmation of APC KO and its effect on the HER2–ERK pathway in APC-KO HeLa cells using Western blotting. ß-Actin served as a loading control. (B) Activity of the Wnt/ß-catenin signaling pathway measured using the TCF/LEF luciferase reporter assay. Nontargeting control cells (NTC) incubated with Wnt3a (100 ng/ml) for 24 h were used as a positive control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.01, *P < 0.05, **P < 0.01 vs NTC cells (n = 3). (C) Measurement of activated RAS (RAS–GTP) using the G-LISA assay. NTC plus Wnt3a (100 ng/ml) was used as a control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.05, *P < 0.05 vs NTC cells (n = 3). (D) Western blot results showing the effects of HER2 WT or HER2 G776S transfection on the HER2 signaling pathway in APC-KO cells. APC-KO cells or NTC cells were transfected with mock or HER2 expression vectors, and the experiments were performed 48 h after transfection. ß-Actin served as a loading control. (E) Measurement of RAS-GTP using the G-LISA assay performed 48 h after HER2 expression vector transfection. The data were standardized to the mean of the activity of NTC cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, *P < 0.05, **P < 0.01 (n = 3). (F) Colony-forming assay results showing the effects of stable transfection with HER2 WT or HER2 G776S in APC-KO cells. The number of colonies was counted in six random low-power fields. Scale bar, 200 µm. Two-way ANOVA: interaction P < 0.01, **P < 0.01.