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. 2014 Mar 14;9(3):e91346. doi: 10.1371/journal.pone.0091346

Figure 1. Downregulation of S1PR3 expression reduces WPB release.

Figure 1

(A) HUVECs were incubated for the indicated time points with 1 μM S1P or SF medium. (B) HUVECs were transfected with a control siRNA SMARTpool (siCTRL) or a siRNA SMARTpool targeting S1PR1 (siS1PR1) or S1PR3 (siS1PR3). Western blot analysis at 72 hours post-transfection showed downregulation of S1PR1 expression or S1PR3 expression. Levels of α-tubulin are shown as a protein loading control. (C,E) siCTRL siS1PR1 and siS1PR3 treated HUVECs were incubated for 45 minutes with 1 μM S1P, 10 μM forskolin (FSK) and 100 μM IBMX, 1 U/ml thrombin (T) or SF medium alone (-). (D,F) siCTRL siS1PR1 and siS1PR3 treated HUVECs were incubated for indicated time points with 1 μM S1P. The amount of VWF secreted in the medium was measured by ELISA. Total amount of VWF secreted is displayed in percentages where the amount of VWF secreted by unstimulated cells t = 45 transfected with siCTRL is set to 100%. Four independent experiments were performed.Statistical significance was assessed by 2-way ANOVA followed by Bonferroni post-hoc test for selected comparison (***P<0.001; **P<0.01 and *P<0.05).