Figure 4
EMMPRIN regulates invasion in oral dysplastic DOK and tumor SCC-9 cell lines in an MMP and uPA dependent manner. A, The in vitro invasive property of DOK and SCC-9 cells incubated with CHO-Emp membranes and treated or not with an anti-EMMPRIN blocking antibody (20 μg/mL) were compared using tissue culture Transwell inserts (8-mm pore size; BD Biosciences) placed in a 24-well culture plate. Cells incubated with CHO control or treated with an anti-IgG antibody were used as control. Cells (1 × 105) suspended in serum-free media were seeded into the upper well of each insert onto membranes coated with growth factor-reduced Matrigel (BD Biosciences). After 48 h incubation, cells that remained in the top compartment were removed by cotton swabs, and cells on the underside of insert filters were fixed, stained, and counted under a microscope. The columns shown represent average values from at least three independent experiments carried out in triplicate; bars, SD. * denotes significant difference with p < 0.05. B, DOK and SCC-9 cells were transfected with EMMPRIN siRNA or scrambled siRNA (Ctl siRNA) prior to invasion assays. The columns represent means of three independent experiments carried out in triplicate; bars, SD. C, Invasive Indices. DOK and SCC-9 cells (1 × 105) seeded into the upper well of tissue culture Transwell inserts coated with growth factor-reduced Matrigel (for invasion assay) or not coated (for migration assay) were incubated with CHO or CHO-Emp membranes (Control and CHO Emp mb respectively). After 48 h of incubation, migrating or invading cells on the underside of insert filters were fixed, stained, and counted under a microscope. Invasion Indices were calculated as percent of the invaded cells relative to the migrated cells. The columns shown represent average values from at least three independent experiments carried out in triplicate; bars, SD. * denotes significant difference with p < 0.05. D, Relative contribution of uPA and MMPs to the in vitro invasive property of DOK and SCC-9 cells. Cells (1 × 105) were seeded into the upper well of matrigel coated inserts and incubated with CHO or CHO-Emp membranes. uPA inhibitor amiloride (20 nmol/L) or MMP inhibitor marimastat (10 μmol/L) were added alone or in combination together with the membranes. After 48 h invading cells were fixed, stained, and counted. Columns represent average values from at least three independent experiments carried out in triplicate; bars, SD. * denotes significant difference with p < 0.05.