E2 box-dependent repression of the CAR promoter and binding of ZEB1 to CAR promoter oligonucleotides and chromatin. A. PANC-1 cells were transfected with CAR promoter/firefly (FF) luciferase constructs (-291/-1) in combination with pRL-SV40 (Promega) encoding renilla (RL) luciferase, an inducible Myc-ZEB1 expression construct, and a "Tet-OFF" plasmid allowing induction of ZEB1 by absence (-Dox), and repression by addition (+Dox) of doxycycline to the culture medium. Cells were lysed forty-eight hours post transfection, and promoter activities were measured with the Dual-Luciferase® Reporter Assay System (Promega). Reporter activities are displayed as fold changes of the FF:RL luciferase RLU ratios relative to the empty vector. Error bars represent standard deviations (biological duplicates). B. In PANC-1 cells ectopically expressed Myc-tagged ZEB1 was precipitated with streptavidin-agarose resin and biotinlyated E-cadherin or CAR promoter oligonucleotides, and then subjected to immunoblotting and detection with an anti-Myc tag antibody. C. Mutations at the E2 boxes in the constructs transfected in A, and in the oligonucleotides used to precipitate ZEB1 (B). E-cadherin promoter mutations are described . D. ChIP assay conducted with PANC-1 cells transiently transfeced with Myc-ZEB1 and stimulated with TGF-β. Myc-ZEB1 was precipitated with an anti-Myc Tag antibody. Co-precipitated DNA was amplified with CAR promoter-specific primers flanking E2 boxes 1 and 2. Shown are SYBR Green I real-time PCR data, normalized to input DNA (prior to precipitation with anti-Myc Tag antibody or control IgG). E. SYBR Green real-time PCR demonstrating overexpression of the total ZEB1 levels in the experiment shown in D. Abbreviations: TCL, total cell lysate; Dox, doxycyline; WT, wild-type; Bx 1, E2 box 1; Bx 2, E2 box 2; Bx 1+2, E2 boxes 1+2; Bx1+3, E2 boxes 1+3 (). p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), n.s.: not significant (Student's t-test: 1-tailed, equal variance).