Figure 3.

LEF1 NAT controls the expression of LEF1 mRNA and protein. (A) Unspliced (+213/−1856) or spliced NAT were synthesized in vitro. A three-fold excess spliced NAT with respect to the unspliced form were transfected in the conditions indicated in Methods. In the control samples an irrelevant RNA (corresponding to a fragment of pcDNA3 plasmid) was transfected. After 36 h, RNA was obtained and levels of LEF1 mRNA were determined using two oligonucleotides corresponding to an amplicon present in the third exon. The results correspond to the average ± range of two experiments performed in duplicate. The asterisk indicates significant (P < 0.05). (B) RWP-1 or HT-29 M6 Snail1 cells were stably transfected with pBabe-LEF1 NAT (unspliced) or pBabe as control. RNA was collected and analyzed by RT-PCR with oligonucleotides specific for LEF1 mRNA, LEF1 NAT (total) or HPRT as control (top panel); alternatively protein extracts were prepared and analyzed by western blot with a polyclonal antibody against LEF1 (Cell Signal) or anti PyrK (Sigma) (bottom panel). (C) The migration capacity of the indicated cell populations was determined as described in Methods. The differences are significant with a P < 0.05.