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

FIGURE 6.

FIGURE 6.

MUC18 overexpression partly rescues PAR1 function, whereas PAFR overexpression completely rescues PAR1 function in C8161-c9 cells. A, Western blot analysis demonstrated stable overexpression of MUC18 in C8161-c9 shPAR1 cells (shPAR1/MUC18) as compared with shPAR1 cells transduced with control empty-vector lentiviral expression plasmid (shPAR1/ev). B, rescuing MUC18 expression in PAR1-silenced cells partly restored the ability of PAR1-silenced cells to attach to HDMECs. Representative images and quantitative results demonstrate that PAR1 silencing down-regulated endothelial cell attachment (shPAR1/ev) as compared with control cell line (shNT/ev). MUC18 overexpression (shPAR1/MUC18) partly restored melanoma cell adhesion to endothelial cells. Data are mean ± S.D. from three experiments performed in triplicates. C, Western blot analysis demonstrated transient overexpression of PAFR in C8161-c9 shPAR1 cells as compared with shPAR1 cells transfected with control empty-vector pcDNA3.1 plasmid. D, rescuing PAFR expression in PAR1-silenced cells restored the ability of PAR1-silenced cells to attach to HDMECs. Representative images and quantitative results demonstrate that PAR1 silencing down-regulated endothelial cell attachment by ∼70% (shPAR1/pcDNA) as compared with control cell line (shNT/pcDNA). PAFR overexpression (shPAR1/pcPAFR) restored melanoma cell adhesion to endothelial cells to 94% of the initial values seen in shNT/pcDNA cells. Data are mean ± S.D. from three experiments performed in triplicates. E, overexpression of MUC18 in PAR1-silenced cells (shPAR/MUC18) did not rescue the loss of melanoma cell retention in the lung after PAR1 silencing (shPAR1/ev). Tumor cell 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. F, overexpression of PAFR in PAR1-silenced cells (shPAR1/pcPAFR) rescued the loss of melanoma cell retention in the lung tissue after PAR1 silencing (shPAR1/pcDNA). Tumor cell 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.