Structure of the CAR promoter. A. Several CXADR (CAR) upstream fragments were PCR-amplified from human genomic DNA and cloned into a firefly (FF) luciferase vector in endogenous constellation, i.e. without vector sequence between the CAR regulatory region, 5'-UTR and the translational start of the luciferase coding sequence. The resulting 5'-deletion series was transfected into PANC-1, MDA-MB-231 and H460 cells, in combination with pRL-SV40 (Promega) encoding renilla (RL) luciferase. Cells were lysed twenty-four 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 (relative light unit) ratios relative to the empty vector. B. Alignment of orthologous CAR promoter sequences and identification of conserved putative ETS and CRE sites [40–42, 49]. A region in which mouse CAR transcripts are likely initiated is indicated (TSS) . Human CAR transcripts may start at around 150 bp upstream of the translational start ATG . C. Wild-type (WT), ETS and CRE mutant -291/-1 CAR promoter constructs were transfected into PANC-1 and MDA-MB-231 cells. Cell lysis and measurements of promoter activities were conducted as in A. Error bars represent standard deviations (biological triplicates). p < 0.001 (***), p < 0.01 (**) (Student's t-test: 1-tailed, equal variance).