(A) Schematic representation of the E-boxes in the promoter region of human Claudin-1 and E-cadherin: open box, E box; +1, putative transcription star point; ORF, open reading frame. (B) Comparison between the 5′-flanking region of human and canine Claudin-1. Alignment of the sequences revealed the presence of conserved E-boxes (underlined). (C) Mutational analyses. The core 5′-CA(G/C)(G/C)TG-3′ sequence of the E-boxes (E1, E2 and E1-2) was mutated to 5′-TG(G/C)(G/C)TG-3′, in various combinations (shadowed boxes). Luciferase reporter constructs carrying wild-type or mutated boxes were transfected in MDCK cells (300 ng), together with (150 ng) Snail or Slug expression constructs or the empty vector (pcDNA3). Luciferase activity in cells co-transfected with wild-type reporter constructs and the pcDNA3 empty vector was defined as 1. Results are means±S.D. for three independent experiments, each performed in quadruplicate. *, P<0.05.