Figure 2.

Zeb2 5′-UTR controls the expression of Luciferase. (A) Diagram of the constructions used in these assays. The sequence corresponding to the Zeb2 promoter (−842/+1) is indicated in black, the 5′-UTR intron (+410/+2928) is indicated in gray, and the rest of the 5′-UTR is indicated in white. The relative position of the oligonucleotides used in this figure is indicated. (B) Firefly Luciferase activity was determined after transfecting the constructions into the indicated cells. Firefly Luciferase was standardized to the value of Renilla Luciferase. (C) RNA was obtained from RWP-1 cells transfected with −842/+2998 construction in pGL3 plasmid. Splicing of exogenous intron was determined by semiquantitative RT–PCR with oligonucleotides specific to the intron and the exon. At this number of cycles, no amplified product was detected in mock-transfected cells. (D) Control or RWP1 cells stably expressing Snail1 cDNA were transfected with the indicated constructions and Firefly Luciferase activity was determined as described. (E) In parallel, RNA was obtained from the above-mentioned transfected RWP-1 cells and levels of exogenous transcript were determined with the indicated oligonuclotides. Results from B and D show the average ± SD of three experiments performed in triplicate.