Expression of DN-RhoK in BBB constructs decreases monocyte migration. TJ alterations in BMVECs after transfection with DA-RhoK or DN-RhoK tagged with c-Myc. Transfection with DN-RhoK was confirmed by c-Myc expression(A) and resulted in the up-regulation of occludin in TJs ([B,D] 2 days after transfection;[F] 7 days after transfection). BMVECs transfected with DA-RhoK show decreased occludin and the loss of intercellular contacts ([C] 2 days after transfection; [E] 7 days after transfection). Double immunolabeling for c-Myc (A) and occludin (B) and immunolabeling for occludin (D-F). Original magnification (A-B) × 400; (C-F) × 200. Images were visualized using a 40 ×/0.75 NA objective and were acquired using a Color View II digital CCD camera. (G) BMVECs in BBB constructs were transfected with DA-RhoK and DN-RhoK. Freshly elutriated monocytes (105) were applied to BBB constructs, and their migration was studied in response to MCP-1 (5 ng/mL in the lower chamber). Transfection of BMVECs with DN-Rho decreased monocyte migration across the BBB by 84%. Results were normalized to migration of uninfected monocytes without MCP-1 (a value of 100%). Values represent the mean of quadruplicate determinations ± SEM.