Individual Rac GTPases Mediate Aspects of Prostate Cancer Cell and Bone Marrow Endothelial Cell Interactions
Effect of Rac depletion on total Rac activation and tumor cell diapedesis across a bone marrow endothelial cell monolayer. (a) Rac isoform expression and isoform-specific depletion in PC-3 cells. Individual siRNAs (1) and (2), specific for Rac1, Rac3, or RhoG, were compared. Messenger RNA was harvested and SYBR green-based qPCR performed using primers specified in Supplemental Table 1. Relative expression levels were normalized to GAPDH expression from the corresponding sample and expressed as arbitrary units (a.u.). (b) is the effect of the RacGEF inhibitor NSC23766 (iRac), siRNA specific for Rac1, Rac3, and RhoG, or scrambled control (siScr) on total Rac activation. Cells were treated with 100 μM NSC23766 for 1 h or 20 μM siRac1, siRac3, or siRhoG. Activation of total Rac was performed using GLISA. Cells treated with iRac or transfected with siRNA to Rac isoforms were compared with untransfected (UT) and representative siRNA-scrambled control (siScr). Each analysis was performed in triplicate with individual transfections. (c) BMECs were layered onto a Matrigel-coated filter and allowed to form a monolayer; 0.5 mL of a suspension of PC-3 cells/mL were added to the BMECs and allowed to undergo diapedesis for 24 h. Treated and transfected cells were compared with untransfected or scrambled controls. (d) Introduction of an RNAi-insensitive Rac3 into siRac3-treated PC-3 cell. Shown in all four panels is the mean ± S.D. of at least triplicate analysis with significance being *. Noncapped lines above the bars represent that the siRNA group is significantly different from controls.
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