ECU formation is extracellular S1P and receptor independent. (A) BM cells derived from WT and SK-1-KO mice were cultured in CS-FCS or complete FCS and ECU numbers determined 7 days after culture. Results show the mean plus or minus SEM for 5 mice per group. *P < .05, compared with WT, 2-tailed paired t test. (B) ECU were counted 4 days after culture in CS-FCS–containing media with administration of S1P (1 μM), pertussis toxin (50 ng/mL), JTE-013 (1 μM), or VPC23019 (10 μM) at initial seeding and every 48 hours thereafter. Results are the mean plus or minus SEM of 3 mice. (C) Semiquantitative RT-PCR was determined for S1P₁ and S1P₂ mRNA expression in BM cells cultured under EPC differentiating conditions for 0 (BM), 3, and 10 days and represents 1 of 5 separate experiments; vertical line(s) have been inserted to indicate repositioned gel lanes. (D) Quantitative RT-PCR was used to determine expression of S1P₁ and S1P₂ in BM cells cultured for 3 and 10 days. Results are the mean plus or minus SEM of receptor expression normalized to the PPIA gene and shown as a ratio over day 3 WT, for 5 separate mice.