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Fig. 1. Snail increases the expression and secretion of MMP-9, which is relieved by Snail RNA interference (siSnail). (A) Identification of the gelatinolytic enzymes produced by MDCK-CMV and MDCK-Snail cells. The cell conditioned media and cellular extracts were collected, centrifuged and 12 µg of protein analysed by zymography in gelatin-embedded SDS polyacrylamide gels. The enzymes were detected as clear bands and a major band of approximately 110 kDa was identified as gelatinase B or MMP-9 by reference to the migration of standard proteins. A conditioned media from MXT-c1.1 (mouse mammary carcinoma) cells was included as control for gelatinase A or MMP-2. (B) Detection of MMP-9 mRNA by RT-PCR. 100 ng of poly(A)⁺ mRNA from MDCK-CMV and MDCK-Snail cells was subjected to RT-PCR. The amplification of cyclophillin was used to normalise for loading (lower panel). (C) Immunofluorescence detection of MMP-9 in MCDK-CMV (left) and MDCK-Snail (right) cells. Note the intense fibrillar and granular staining in MDCK-Snail cells. (D) Cells collected from subconfluent cultures of MDCK-CMV and MDCK-Snail cells were lysed, and the expression of MMP-9 was analysed by western blotting using an anti-MMP-9 polyclonal antibody. -Tubulin was used as loading control. (E) Snail silencing blocks the induction of MMP-9 expression. Upper four panels: RT-PCR analysis of MMP-9, Snail and E-cadherin mRNA levels in MDCK-CMV, MDCK-Snail and in three independent stable clones (C1, C3 and C4) generated after siSnail transfection (MDCK-siSnail) in MDCK-Snail cells. GAPDH mRNA levels are shown as loading control. Lower panel: the effects of Snail interference on secreted MMP-9 were analysed by zymography in gelatin-embedded SDS polyacrylamide gels.

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