SK-1 mediates the Ang-1 effects on permeability. (A) HUVECs were transfected with control siRNA or siRNA against hSK-1. After 48 hours, cells were treated with or without Ang-1 for 30 minutes and SK-1 activity measured. Pooled data from 3 experiments are shown and are expressed as the fold change in relation to untreated control cells where the SK-1 activity was set to 1.0 (*P < .001 vs control siRNA cells without treatment). NS indicates no significant difference versus SK-1 siRNA cells without treatment. (B) HUVECs were transfected with control siRNA or siRNA against hSK-1. Forty-eight hours later, cells were untreated or treated with Ang-1 (0.2 μg/mL) for 30 minutes, and permeability is measured. Permeability is given as the FITC-dextran passed after 30 minutes. Pooled data from 3 experiments are shown (mean ± SEM; *P < .01 vs control siRNA cells without treatment). NS, vs SK-1 siRNA cells without treatment. (C) HUVECs were transfected with control siRNA (Ci and Cii) or siRNA against hSK-1 (Ciii and Civ). Forty-eight hours later, cells were untreated (Ci and Ciii) or treated (Cii and Civ) with Ang-1 for 1 hour and then stained for VE-cadherin. In panel Ci, arrow shows diffuse, broad VE-cadherin staining with classic zipper-like pattern indicative of immature junctions. In panel Cii, arrow indicates increased VE-cadherin staining at the junctions, with a more linear staining pattern indicative of mature EC junctions. (D) HUVECs were transfected with control siRNA or siRNA against hSK-1. Forty-eight hours later, cells were untreated (−) or treated (+) with Ang-1 (0.2 μg/mL) for 30 minutes then were lysed and immunoprecipitated with an anti-Tie2 antibody. Western blots for phosphotyrosine (top panel) and Tie2 (bottom panel) are shown. For imaging information, see “VE-cadherin staining” section in “Methods.”