FIGURE 2.

Snail1 controls Zeb1 mRNA levels. Panels A and B, RNAs were prepared from the indicated cell lines stably transfected with Snail1-HA or infected with a retrovirus expressing human SNAIL1 shRNA (hshRNA1) or scrambled control shRNA. Expression of the indicated genes was determined by qRT-PCR. Values are presented as fold-stimulation by Snail1 with respect to control cells (transfected with the empty plasmid) (A) or as percentage of expression with respect to cells infected with the control shRNA (B). 45 and 55% inhibition of Zeb1 expression was obtained with another human SNAIL1 shRNA (hshRNA2) in MiaPaca-2 and SW-620, respectively. Panel C, activity of the 1004/+29 ZEB1 promoter was determined after transfection of the indicated cell lines stably expressing Snail1-HA or treated with TGF-β for 48 h. Triplicates were systematically included and experiments were repeated at least three times. The figure shows the average ± S.D. of 3–5 experiments. Values obtained for Snail1-expressing cells were different from the corresponding control with a p < 0.01 in RWP1 cells and p < 0.05 in SW-480 and NMuMG cells; differences between NMuMG and TGF-β-treated NMuMG cells were also statistically different (p < 0.05). Panels D and E, RNA was prepared from RWP1, RWP1-Snail1 (D), or NMuMG cells treated 1 or 24 h with TGF-β (E). When indicated, Snail1 expression was inhibited in NMuMG cells using a specific siRNA (msiRNA). Expression of miR-200 was determined as indicated under “Experimental Procedures.” Absence of amplification of DNA was verified as control. Analysis of Pumilio RNA was carried out to determine that equal amounts of RNA were used.