Fig. 7

Frz8 was required for the RNF43-induced ubiquitination of phosphorylated E-cadherin. a, Immunoblot analysis of Frz8, Frz2, Dishevelled 3 (Dvl3), LGR6, and β-actin in A549 and H522 cells. b, A549 and H522 cells stably transfected with control shRNA or RNF43 shRNA (RNF43 KD) via adenovirus were seeded overnight. The total cell lysates (TCLs) were subjected to immunoprecipitation (IP) using an anti-Frz8 antibody. Ubiquitinated Frz8 was detected by western blot with an anti-ubiquitin antibody (upper blot). Frz8 and RNF43 in the TCLs were detected by western blot (lower blot). c, A549 cells stably transfected with control shRNA or Frz8 shRNA (Frz8 KD) via lentivirus were seeded overnight. The TCLs were subjected to IP using an anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected by western blot with an anti-ubiquitin antibody (upper blot). E-cadherin, Frz8, and β-actin in the TCLs were detected by western blot (lower blot). d, Schematic representation of reporter plasmids containing full-length RNF43 and two truncated versions of RNF43 as shown. e, RNF43-knockdown A549 cells stably transfected with RNF43, RNF43-R, or RNF43-P via adenovirus were seeded overnight. The TCLs were subjected to IP using anti-E-cadherin and anti-HA antibodies. Ubiquitinated E-cadherin, E-cadherin, Frz8, and HA were detected by western blot (upper and middle blot). E-cadherin and β-actin in the TCLs were detected by western blot (lower blot). f, A549 cells treated with DMSO (Control) or monoclonal antibodies (10.0 mmol/L) against the cysteine-rich domain (CRD) of Frz8 and the RNF43 protease domain (PA) were seeded overnight. The TCLs were subjected to IP using an anti-E-cadherin antibody. Ubiquitinated E-cadherin, RNF43, and E-cadherin were detected by western blot (upper blot). E-cadherin, Vimentin, and β-actin in the TCLs were detected by western blot (middle blot). β-catenin was detected in the nuclear extract (lower blot). g, A549 cells treated with DMSO (Control) or monoclonal antibodies (10.0 mmol/L) against the cysteine-rich domain (CRD) of Frz8 and the RNF43 protease domain (PA) were seeded overnight. The TCLs were subjected to IP using an anti-RNF43 antibody. Frz8, RNF43, and E-cadherin were detected by western blot. h, EMT morphological changes and metastatic incidences in A549 cells treated with DMSO (Control) or monoclonal antibodies (10.0 mmol/L) against the cysteine-rich domain (CRD) of Frz8 and the RNF43 protease domain (PA) (χ² test, *, P < 0.05). Scale bars, 50 μm. i, A549 cells treated with DMSO (Control) and RSOP1–4 were seeded overnight. The TCLs were subjected to IP using anti-E-cadherin and anti-Frz8 antibodies. Ubiquitinated E-cadherin and RNF43 were detected by western blot (upper and middle blot). E-cadherin and β-actin in the TCLs were detected by western blot (lower blot)