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. 2021 Dec 22;13(4):1276–1296. doi: 10.1016/j.jcmgh.2021.12.014

Figure 3.

Figure 3

β-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.