Effect of 5-aza-dC treatment with β-catenin and/or E-cadherin depletion.

<p>After knockdown of β-catenin (A) or E-cadherin (C), protein expression of SPC and A549 cell lines were examined 48 h after 5-aza-dC treatment using the indicated antibodies. (B, D) Graphs show the quantification of protein expression after 5-aza-dC treatment and siRNA transfection. GAPDH was used as a control. (E) Representative images of cells on the bottom of the Transwell membranes show the changes in invasive cell numbers (×400, Scale bar = 50 µm). (F) Number of cells invading onto the lower surface of the filter was counted, each carried out in triplicate. (Bars represent SD. *<i>P</i><0.05, compared to the control). (G) Dual-luciferase assay results show that both 5-aza-dC treatment and β-catenin depletion resulted in no significant changes in Wnt signaling activities compared with Wnt3a treated cells. Each of the experiments was repeated in triplicate. (H) Immunofluorescent staining showing that the membranous β-catenin is enhanced by 5-aza-dC treatment only in SPC cell line, but not in A549 cell line. Depletion of β-catenin attenuates membranous β-catenin in both SPC and A549 cell lines, but depletion of E-cadherin has no effect on membranous expression of β-catenin in either SPC or A549 cell line. Treatment with 5-aza-dC slightly altered the amounts of membranous β-catenin expression after β-catenin or E-cadherin depletion in SPC cell line but not in A549 cell line. (Scale bar = 20 µm, β-catenin antibody was replaced by normal mouse IgG as a negative control).