Figure 3. MT3-MMP silencing inhibits fibrin invasion and enhances collagen invasion of WM852 cells.

(A) MT1-MMP and MT3-MMP mRNA expression in WM852 cells transfected with control siRNA (Ctrl) or siRNAs targeting MT1-MMP (siMT1) or MT3-MMP (siMT3-1 and siMT3-2) were quantified by qPCR (n = 3). (B and C) The protein levels of MT1-MMP (B) and MT3-MMP (C) were assessed by immunoblotting after transient transfection of the siRNAs or MT3-MMP cDNA (MT3-MMP) as indicated. MT1-MMP was detected as a 60 kDa band and MT3-MMP as 65/60 kDa bands that were downregulated by the corresponding siRNAs. An additional MT3-MMP fragment of 37 kDa in size was detected in the cells after transient transfection with MT3-MMP cDNA. Ponceau-staining served as a loading control. Asterix marks a non-specific band. (D) Cell surface MT3-MMP was detected by surface biotinylation of cells transfected with control siRNA or the most efficient MT3-MMP siRNA (siMT3-1), followed by immunoprecipitation. Asterix marks a non-specific band. (E) Light micrographs of fibrin and collagen cross-sections visualize the invasion of WM852 cells transfected with the indicated siRNAs. The cells were plated atop 3D fibrin and type I collagen 1 d after siRNA transfections and allowed to invade for 5 d. Quantitative results are expressed as the number of invasive foci per microscopic field (n = 3).