PKP3 is partially relocalized to the plasma membrane upon ZEB1 knock-down. For immunofluorescence microscopy MDA-MB-231 cells were treated with ZEB1-specific (si-ZEB1) or unspecific scrambled (si-Control) si-RNAs for three days, fixed with formaldehyde and stained with PKP3, E-cadherin, Desmoplakin and ZO1 specific antibodies. Bars, 10 μm.

ZEB 1 represses the activity of the mouse PKP3 promoter. (A) Scheme of the first four kbp of the mouse PKP3 promoter. E-boxes are indicated by black bars. The position of the four promoter fragments are shown below the scheme (mPKP3-prom1-4). (B) Reporter assays were performed as indicated in Fig. 3 using a mouse full-length ZEB1 expression vector and EpH4 murine mammary epithelial cells.

Knock down of ZEB1 causes upregulation of PKP3 expression. (A) RNAi-mediated knock down of ZEB1 in MDA-MB-231 breast cancer cells. Three days after treatment with either ZEB1-specific (si-ZEB1) or unspecific scrambled (si-Control) siRNAs total RNA was harvested and processed for Affymetrix Gene Chip analysis™. Diagram shows relative-change of mRNA levels of ZEB1 and PKP3 after ZEB1-specific knock down compared to cells treated with scrambled siRNA (si-Control). (B) PKP3 and E-cadherin mRNA levels after ZEB1 knock down. mRNA levels were analysed by semi-quantitative RT-PCR three days after ZEB1 knock down (see A). Actin was included for normalization. (C) Protein levels of PKP3, E-cadherin and ZEB1 after knock-down of ZEB1. For immunoblotting cells were treated with siRNAs as indicated in (A). Total MDA-MB-231 cell lysates were separated by SDS-PAGE and immunoblots were performed using antibodies to ZEB1, PKP3, E-cadherin and Actin (loading control).

ZEB1 directly associates with the PKP3 promoter and represses its activity. (A) Scheme of the human PKP3 promoter. Relative positions of E-boxes are depicted as black bars along the first four kbp of the human PKP3 promoter. (B) The two proximal E-boxes (shaded) close to the transcription initiation site are conserved in the human and mouse PKP3 promoter. (C) ZEB1 physically interacts with the human PKP3 promoter. ZEB1 associated chromatin fragments derived from MDA-MB-231 cells were pulled down in chromatin immunoprecipitation (ChIP) experiments. PKP3 promoter regions were amplified by PCR (see ChIP1 and ChIP2 in A). Input confirms equal chromatin loading. Lane ZEB1: ZEB1-specific antibody; Lane Control: unspecific goat antibody. (D) ZEB1 represses the human PKP3 promoter activity. Human ZEB1 or Snail1 expression vector or control vector (p120ctn) were transfected into human epithelial MCF7 cells together with one of the two human PKP3 promoter constructs depicted in (A) (hPKP3-prom1 and hPKP3-prom2). Relative repression by ZEB1 and Snail1 (Sna1) is shown by mean values of four independent measurements compared to control vector. Bars represent standard errors on the mean.

ZEB1 and PKP3 localization in human colon cancer. (A) Colon cancer specimens were double-stained for ZEB1 (brown stain) and PKP3 (violet stain). Left panel, overview of differentiated bulk tumour area. Right panel, higher magnification of differentiated area. PKP3 is expressed in differentiated regions of the tumour (violet stain, arrows), whereas ZEB1 is mostly detected in tumour-associated stroma cells (brown stain, arrowheads). (B) Double staining of ZEB1 (brown) and Smooth Muscle Actin (violet). Note that some of the activated tumour-associated fibroblasts express nuclear ZEB1. (C) Invasive area of colon tumour stained with Hematoxylin/Eosin. (D) Upregulation of ZEB1 in invasive cancer cells at the tumour–host interface (brown stain, arrowheads). ZEB1 expression is accompanied by downregulation of PKP3 (violet stain in cells marked with arrowheads). Note that residual PKP3-specific staining is still detectable in the cytoplasm of ZEB1 expressing cells. Bars, 120 μm (A, B, C left) or 30 μm (A, B, C, right and D).