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. 2009 Aug 24;284(42):28845–28855. doi: 10.1074/jbc.M109.042150

FIGURE 5.

FIGURE 5.

Silencing PAR1, PAFR, or MUC18 in C8161-c9 cells decreased their adhesion to HDMECs, diapedesis, and retention in mouse lungs. A, Western blot analysis demonstrates down-regulation of PAFR or MUC18 proteins in C8161-c9 cells transduced with lentiviral PAFR-targeting shRNA (shPAFR) or MUC18-targeting shRNA (shMUC18). B, silencing PAR1, PAFR, or MUC18 in C8161-c9 cells decreased their adhesion to HDMECs. Representative images and quantitative results (bar graphs) demonstrate that PAR1, PAFR, or MUC18 silencing down-regulated endothelial cell attachment by 69%, 84%, and 62%, respectively. C, representative ECIS plots show a decrease in impedance of HDMEC monolayers on the addition of control C8161-c9 shNT cells (shNT), indicating transendothelial migration of melanoma cells (blue lines in each panel). Arrows indicate the addition of melanoma cells to HDMECs. PAR1 (shPAR1), PAFR (shPAFR), or MUC18 (shMUC18) silencing inhibited transendothelial cell migration (green lines). The impedance in samples with HDMECs supplemented with media alone remained stable during the entire experiment (red lines). D, PAR1, PAFR, or MUC18 silencing inhibited melanoma cell retention in the lung tissue of nude mice by 89%, 61%, and 79%, respectively. Tumor cells retention was measured 24 h after tail-vein injection and is expressed as the amount of radioactivity retained per gram of tissue. *, p < 0.01.