Fig. 9. MAPK and PI3K signalling pathways are involved in the induction of MMP-9 proximal promoter by Snail. (A) MMP-9 promoter activity was measured in MDCK-CMV cells by using the reporter construct pMMP9-389luc in the absence or presence of the Snail expression vector, and in the presence or absence of the following inhibitors: UO126 (10 µM); wortmannin (40 nM); SB 203580 (10 µM) or JNK inhibitor II (100 nM). Control cells (untreated) were unstimulated and treated with DMSO alone. Promoter activity is expressed relative to the value obtained in MDCK-CMV untreated cells and results are expressed as the mean±s.d. of three independent experiments. (B,C) Effect of Snail on Erk (B) and Akt (C) phosphorylation. Total cellular protein was extracted from MDCK-CMV and MDCK-Snail untreated cells and cells treated with wortmannin (C). 150 µg were analysed by western blotting using specific antibodies for the phosphorylated forms of these protein kinases. -tubulin was used as a loading control. (D) Cell extracts (30 µg) from MDCK-CMV and MDCK-Snail cells were used in western blots to determine Sp-1 phosphorylation status using a specific antibody that recognizes the phosphorylated and unphosphorylated isoforms. -tubulin was used as a loading control. One representative experiment out of five is shown. (E) Nuclear extracts of MDCK-Snail cells cultured for 24 hours in serum-free medium were analysed by EMSA after transient transfection with MEK-DN expression vector or empty plasmid. The ³²P-labelled wild-type (wt) oligonucleotide encompassing the sequence from nts -97 to -76 is the same as that in Fig. 7B. The same nuclear extracts were analysed by western blotting using specific antibodies for the phosphorylated forms of Erk protein kinases. Anti-total Erk antiserum was used in the same membrane as a loading control.