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. 2012 Sep 19;287(46):38716–38728. doi: 10.1074/jbc.M112.373159

FIGURE 1.

FIGURE 1.

Identification of MMP-13. A, schema shows the strategy to identify MMP-13. Periostin, IFITM1, and Wnt-5b are identified as the invasion-related molecules by comparing the gene expression profile between the parent (MSCC-1 cells) and a highly invasive clone (MSCC-Inv1 cells). To identify common up-regulated genes, we compared the gene expression profiles of control versus periostin-overexpressing cells, control versus IFITM1-overexpressing cells, and control versus Wnt-5b-overexpressing cells. MMP-13 is commonly up-regulated among periostin-, IFITM1-, and Wnt-5b-overexpressing cells. B, expression of MMP-13 in HNSCC cell lines. Expression of MMP-13 mRNA in six HNSCC cell lines: HSC2, HSC3, HSC4, Ca-9-22, Ho-1-N-1, and Ho-1-U-1 was examined by RT-PCR. GAPDH was used as a loading control. C, generation of MMP-13-overexpressing cells. pcDNA3.1-FLAG-MMP13 was transfected into HSC3 cells. After selection, we obtained four stable clones and one stable pool clone of MMP-13-overexpressing cells. Ectopic expression of MMP-13 was examined by immunoblotting with an anti-FLAG antibody. In further experiments, clone 1 was used. D, MMP-13 ability was determined by a MMP-13 inhibitor assay kit as described under “Experimental Procedures.” Conditioned medium was collected from control HSC3, MMP-13-overexpressing HSC3, control Ho-1-N-1, MMP-13 siRNA-treated Ho-1-N-1, control HSC4, and MMP-13 siRNA-treated HSC4 cells. The reaction was initiated by adding 100 μl of substrate solution, and the reaction fluorescence intensity was determined at λemission = 450 nm and λexcitation = 345 nm. The graph shows fluorescence intensity. All assays were carried out in three replications. E, ectopic expression of FLAG-MMP-13 was examined by immunoblotting with an anti-FLAG antibody. β-Actin expression was used as a loading control. Expression of MMP-13 in condensed conditioned medium was detected by immunoblotting with an anti-MMP-13 antibody. F, MMP-13 knockdown in HSC4 and Ho-1-N-1 cells. MMP-13 siRNA was transfected into HSC4 and Ho-1-N-1 cells. A scrambled sequence that does not show significant homology to rat, mouse, or human gene sequences was used as a control. After 48 h, cells were collected, and MMP-13 expression was examined by Western blot (WB) analysis. β-Actin expression was used as a loading control.