Fig. 2.

a Antagonistic effects of KRO-105714 by SPC (a: [³⁵S]-GTPγS binding assay, b: Migration assay). The HEK-293 cells stably expressing hS1P1 were homogenized with assay buffer and pre-incubated with GTP-binding buffer in the presence or absence of 0.8 nM, 4 nM, 20 nM, 100 nM, 500 nM and 2.5 μM KRO-105714 with 10 μM SPC for 30 min. 1200 Ci/mmol; 0.1 nM [³⁵S]-GTPγS was loaded and incubated for 30 min. Nonspecific binding was determined in the presence of 30 μM GTPγS. Each dose of the test compound was assayed in triplicate on pooled samples. b Migration assays were performed in Transwell 96-well chambers. SPC (10 μM) was placed in the lower well. HUVECs were harvested and treated with test compounds for 30 min before seeding. The cell suspension was loaded into the upper well. Cells were allowed to migrate for 4 h in a humidified chamber. The cells were fixed and stained with hematoxylin and eosin. Cell migration was quantified by counting the cells that attached to the lower surface of the filter using an optical microscope at X200 magnification. Fifteen fields were counted for each assay. Each dose of the test compound was assayed in triplicate on pooled samples. c Tube formation effects of KRO-105714 in HUVECs. Representative photographs of HUVEC tube formation induced by control (vehicle, 0.1% DMSO) (a), 10 μM of SPC (b), 10 μM of SPC, and 2.5 μM of KRO-105714 are shown from three different independent experiments