Repression of inflammatory marker gene expression by DSCR1 on activated endothelial cells. (A) Real-time RT-PCR analysis of HUVECs transduced with adenoviral vectors (MOI = 100) encoding for DSCR1 (Ad-DSCR1-FLAG) or control LacZ (Ad-LacZ). Two days after transduction, cells were incubated for 5 to 6 hours with growth medium alone or with growth medium complemented with PMA (200 ng/mL) and IO (5 μM), VEGF (30 ng/mL), thapsigargin (50 nM), or a combination thereof as indicated. Total RNA was isolated and levels for Cox-2 (i), E-selectin (ii), and TF (iii) were analyzed by real-time RT-PCR analysis. Data shown represent the average ± SD of triplicate samples of one representative of total 3 independent experiments. RRUs are calculated as described by Gerber et al³  by using GAPDH as reference gene. (B) FACS analysis of transiently transfected endothelial cells stimulated by various compounds. CsA (1 μM) was added to transduced cells 2 hours prior to stimulating cells with either PBS (NA), PMA (200 ng/mL), IO (5 μM), TNF-α (10 ng/mL), or a combination thereof. At 24 hours after stimulation, cells were removed from the culture dish by incubation in 25 mM EDTA (ethylenediaminetetraacetic acid/PBS for 10 minutes. Cells were analyzed for expression of E-selectin, VCAM-1, TF, and ICAM-1. White areas under the chromatograms represent expression levels on cells transduced with Ad-DSCR1; gray areas represent expression levels on LacZ-transduced cells. Data shown are from one representative of total 3 independent experiments. (C) Western blotting analysis of total cell extracts of HUVECs for Cox-2 and TF, respectively. To control for equal loading, an antibody recognizing γ-adaptin was used. Data shown are from one representative experiment of 3 independent experiments.