Fig. 5

RNF43-induced E-cadherin ubiquitination and degradation initiated EMT phenotype via allowing β-catenin to translocate into the nucleus in a c-Src-dependent manner. a, A549 cells were stably transfected with the lentiviral control shRNA, RNF43 shRNA, or c-Src shRNA. Snail, Slug, Twist, E-cadherin (E-cad), Vimentin, Fibronectin, N-cadherin (N-cad), and β-actin in total cell lysates (TCLs) were detected by western blot. b, A549 cells stably transfected with lentiviral control shRNA, RNF43 shRNA, or c-Src shRNA were seeded overnight. TCLs were subjected to immunoprecipitation (IP) using an anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected by western blot with an anti-ubiquitin antibody (upper blot). Phospho-β-catenin (p-β-catenin), β-catenin, GSK-3β, RNF43, and β-actin in TCLs were detected by western blot (middle blot). β-catenin was detected in the nuclear extract of A549 cells (lower blot). c, The TCLs of A549 cells stably transfected with lentiviral control shRNA, RNF43 shRNA, or c-Src shRNA were subjected to IP using an anti-β-catenin antibody. E-cadherin, GSK-3β, TCF-4, and β-catenin were detected by western blot. d, A549 cells were treated with an shRNA either not targeting TCF-4 (Control) or targeting TCF-4 (TCF-4). The in vitro β-catenin antibody 7A7 was delivered into A549 cells. Immunoblot analysis of LaminA/C (loading control), TCF-4, E-cad, Vimentin, and nuclear/total β-catenin was performed using appropriate antibodies. e, A549 cells were treated with an shRNA either not targeting (Control) or targeting TCF-4 (TCF-4). The in vitro β-catenin antibody 7A7 was delivered into A549 cells. The mRNA levels of E-cad, Vimentin, and RNF43 were examined by RT-PCR. GAPDH mRNA was used to confirm equal loading. f and g, β-catenin and TCF-4 chromatin immunoprecipitation (ChIP) in A549 cells. PCR was carried out using primers specific for the promoter regions of E-cad, Vimentin, and RNF43. One-fifth of the input DNA from each sample was also amplified and designated as Input. h, Schematic representation of reporter plasmids containing the control vector, E-cadherin promoter, Vimentin promoter, Vimentin promoter with WRE deletion (Vim mut), RNF43 promoter, and RNF43 promoter with WRE deletion (RNF43 mut). i, A549 cells were transiently transfected with the reporter plasmids described in f. Luciferase activities were measured in triplicate (mean ± SD, t test, *, P < 0.05)