Figure 4. IL-32α overexpression downregulates E-cadherin expression and induces F-actin polymerization.

A. G361-vector and G361-IL-32α cell lines were detached using enzyme-free dissociation buffer. Flow cytometry assays were performed using the PE-conjugated mouse anti-human E-cadherin antibody. B. E-cadherin, β-catenin, phospho-β-catenin and GSK-3β expression was evaluated in G361-vector and G361-IL-32α cell lines. C. Total RNA was isolated from G361-vector and G361-IL-32α cells. After reverse transcription, PCR was performed with primers for β-catenin or β-actin. D. G361-vector and G361-IL-32α cells were attached to coverslips then fixed and permeabilized as described in the Materials and Methods. After permeabilization, the coverslips were blocked with 1% BSA in PBS for 1 hour and incubated at 4°C overnight with rabbit anti-human β-catenin antibody. Coverslips were then incubated with FITC-conjugated goat anti-rabbit IgG antibody. A laser scanning confocal microscope was used for analyses. E. G361-vector and G361-IL-32α cells were incubated on coverslips. Cells attached to the coverslips were fixed and permeabilized as mentioned in Materials and Methods. F-actin staining was performed using phalloidin-conjugated Alexa Fluor 647. Confocal microscopy assays were performed as described. These data represent one of three independent experiments.