Fig. 8.

Dkk1 downregulates transcriptional active β-catenin. A: Western blot analysis of active β-catenin (ABC dephosphorylated at Ser³⁷/Thr⁴¹) and total β-catenin expression in whole cell lysates of EPC2 cells following treatment with rhDkk1. B: Western blot analysis of ABC protein following efficient Dkk1 short interfering RNA (siRNA)-mediated knockdown. β-Actin served as loading control. Blots represent results from 3 experiments. *P ≤ 0.05. C: ELISA of Dkk1 secretion in culture medium following transfection with Dkk1 siRNA. Values are means ± SE of 3 independent biological replicates. *P ≤ 0.05 vs. control siRNA. D: quantitative RT-PCR analysis of mRNA levels for Dkk1, Axin2, Ki-67, and p16 genes in EPC2 cells following Dkk1 siRNA-mediated gene silencing. Gene expression was normalized to β-actin expression. Values (means ± SE of 3 biological replicates) are relative to mock control. *P ≤ 0.05, #P ≤ 0.01. &Dkk1 knockdown ≥80%. E: immunofluorescence microscopy for colocalization of Dkk1 and ABC protein in esophageal mucosa. Arrowheads, basal layer of esophageal epithelium. Images represent results from ≥3 different tissues.