Inhibition of pro-uPA activation on THP-1 cells by siRNA-mediated down-regulation of matriptase expression. (A) THP-1 cells were transiently transfected with 100 nM nontargeting siRNA (matscram), each matriptase specific siRNA (mat162 or mat1725), or both matriptase specific siRNAs in combination (162 + 1725). Twenty-four hours after transfection, RNA was isolated, reverse transcribed, and subjected to qRT-PCR. Values are corrected for 18S ribosomal RNA levels and expressed as a percentage of matriptase mRNA in nontransfected cells. Data are shown as mean ± SD (n = 3). None of the siRNAs used affected expression levels of uPA, uPAR, HAI-1, PAI-1, or PAI-2 as determined by qRT-PCR. (B) Representative cell surface plasminogen activation on control THP-1 cells transfected with nontargeting siRNA (▪) compared with cells transfected with mat162 + 1725 (○, □), representing data from 3 independent transfection experiments. The 2 plasmin generation curves shown for targeted cells (○, □) are fitted using 15% and 0% initial activation of pro-uPA, respectively. (C) Data from these plasminogen activation experiments and similar experiments in which pro-uPA activation by the cells was determined directly using H-Glu-Gly-Arg-AMC (as in Figure 2). Data are shown as mean ± SD (n = 3) for nontargeting (dark bars) and targeting (light bars) siRNA and are expressed as a percentage of the pro-uPA activation observed in nontransfected cells. Similar data were obtained when each of the targeting siRNAs was transfected individually.