Fig. 3.

Endogenous regulation of ZEB1 by β-catenin/TCF4. (A) Endogenous β-catenin and TCF4 regulate endogenous ZEB1 mRNA. SW480 cells were transfected with 50–100 nM of a control siRNA or specific siRNAs for ZEB1, β-catenin, and TCF4. Relative mRNA levels were determined by qRT-PCR respect to GAPDH. (B) Endogenous β-catenin and TCF4 regulate endogenous ZEB1 protein. SW480 cells were transiently transfected with control and specific siRNAs and cell lysates were immunoblotted for ZEB1 (H-102), β-catenin (clone 14), TCF4 (H-125), as well as α-tubulin (B5-1-2) to control for loading. (C) Nuclear accumulation of β-catenin induces endogenous ZEB1. (Left) HTC116 cells were transfected with 0.5 μg of ZEB1 promoter and, after 48 h, treated with 10 ng/mL of LMB for different periods before cell lysates were processed for luciferase activity quantification (see Supporting Information). (Center) HCT116 cells, treated with 10 ng/mL of LMB for different periods, were assessed for ZEB1 mRNA levels by qRT-PCR. (Right) HCT116 cells, treated with 10 ng/mL of LMB for different periods, were lysed, and protein levels were determined by Western blot with ZEB1 (H-102) and α-tubulin (B5-1-2) Abs.