S1P prevents thalidomide-induced growth inhibition, ceramide generation through nSMase, and depletion of VEGF receptor expressions (neuropilin-1, neuropilin-2, and FLK-1) in HUVECs. (A-D) Human umbilical vein endothelial cells (HUVECs) were pretreated without or with 0.1 μM S1P for 1 hour before 200 μM thalidomide, and then harvested at the indicated times (A,B) or 6 hours after thalidomide treatment (C,D). (A-B) ▪ indicates 200 μM thalidomide; ♦, 200 μM thalidomide plus 100 nM S1P; and ○, control. Pretreatment with S1P restores thalidomide-induced inhibition of cell growth (A), generation of ceramide (B), and activation of nSMase (C) to the control levels. S1P also restores thalidomide-inhibited mRNA levels of FLK-1, neuropilin-1, and neuropilin-2 in HUVECs. (D) The expression levels of FLK-1, neuropilin-1, and neuropilin-2 were measured by the RT-PCR–based amplification of mRNA as described in “Materials and methods.” The results are obtained from 3 independent experiments (A-C) or the representative of 3 different experiments (D). The error bars indicate 1 SD. *Statistically significant difference between pretreatment with and without S1P (P < .01).