Figure 3

EMMPRIN regulates uPA in oral dysplastic DOK and tumor SCC-9 cell lines. A, (a) CHO cells were transfected with EMMPRIN cDNA as previously described [18]. Membranes were isolated from CHO-Emp and CHO control cells (CHO-Emp Mb and CHO Mb, respectively) by differential centrifugation and 10 μg of membrane extract were analyzed for EMMPRIN content by immunoblotting. (b) DOK and SCC-9 cells (80% confluence) were incubated with 20 μg/mL CHO Mb or CHO-Emp Mb in serum-free medium for 24 h and the conditioned medium was analyzed for uPA activity using casein-plasminogen zymography and for gelatinase activities of MMP-2 and MMP-9 using gelatin zymography (one representative zymography of three independent experiments). (c) EMMPRIN, uPA, MMP-2, MMP-9 and TIMP-1 transcripts were quantified using quantitative RT-PCR in DOK and SCC-9 cells incubated for 24 h with CHO Mb or CHO-Emp Mb (20 μg/mL). Columns are means of gene expression relative to TBP housekeeping gene of at least three independent experiments carried out in triplicate; bars , SD. * denotes significant difference with p < 0.05. (d) SCC-9 and DOK cells were grown in microscope slide chambers to 50-60% confluence and incubated with CHO Mb or CHO-Emp Mb (20 μg/mL). After 24 h, cells were immunostained for uPA using a goat anti-human antibody (green) and counterstained with DAPI (blue). A more intense staining was observed in CHO-Emp mb treated DOK and SCC-9 cells compared to the control CHO mb treated cells. B, SCC-9 and DOK cells were incubated with 20 μg/mL of an anti-EMMPRIN blocking antibody (Ancell, Bayport, MN) or with a non-immune IgG antibody in serum-free medium. After 24 h incubation, conditioned medium was harvested for gelatinase and uPA casein zymography. Columns represent average values of the densitometric quantification from at least three independent experiments carried out in triplicate; bars , SD. * denotes significant difference with p < 0.05. C, DOK and SCC-9 cells were transfected with EMMPRIN siRNA (Emp-siRNA) or scrambled control siRNA (Ctl siRNA) at 33 nmol/L concentration. EMMPRIN and uPA mRNA expression was evaluated by quantitative RT-PCR analyses. Columns represent mean ± SD of relative expression to TBP housekeeping gene of at least 3 independent experiments carried out in triplicate; bars, SD. * denotes significant difference with p < 0.05.