Figure 3: Agonists (a-c) and inhibitors (d-f) of S1P₁ regulate binding, association and transendothelial transport of HDL in human aortic endothelial cells (HAECs).

HAECs were cultured for 72h. Cells were then treated with S1P₁ agonist (SEW2871, 20nM; A-C) or S1P₁ inhibitor (W146, 20nM, D-F) for 30 minutes, at 37 °C as indicated. To study cellular binding, association and transport, HAECs were incubated with 10μg/mL of ¹²⁵I-HDL for 1 hour in the absence (total) or in the presence of 40-fold excess of unlabeled HDL, to record nonspecific interactions. Specific binding, association and transport were calculated by subtracting nonspecific values from total values. Specific binding was measured by incubating cells with ¹²⁵I-HDL (A, D) at 4°C. To measure specific cell association, cells were incubated with ¹²⁵I-HDL (B, E) at 37°C. For the measurement of transport, HAECs were cultured on inserts. The transport of ¹²⁵I-HDL (C, F) from the apical to basolateral compartment was measured at 37°C. The results are presented as means ± SEM of three independent triplicate experiments (n=3). P was calculated by unpaired Mann Whitney U-test.