Figure 2.

SK1 overexpression causes intra- and extracellular increase of S1P in HEK293 cells. A) Stable HEK cells transfected with vector (HEK-V) or SK1 (HEK-SK1) were subjected to mRNA extraction and real-time PCR analysis for hSK1. Real-time PCR data are expressed as mean ± sd normalized expression of 3 independent mRNA extractions, using β-actin as reference gene. B) HEK-V and HEK-SK1 were collected for Western blot. Membranes were probed with the following antibodies: hSK1 (1:500) and actin (1:10,000). Blot is representative of 2 independent experiments performed in duplicate. C, D) HEK-V (C) and HEK-SK1 cells (D), incubated for 2 h in trapping medium (described in Materials and Methods), were collected and analyzed by mass spectrometry for intracellular and extracellular S1P content. Results are means ± sd (pmol/mg protein) of 3 independent experiments performed in duplicate. E, F) HEK-V (E) and HEK-SK1 cells (F), incubated for 2 h in trapping medium, were pulsed for 10 min with C₁₇-d-erythro-sphingosine to a final concentration of 1 μM. C₁₇-S1P was measured in the cells and in the medium by mass spectrometry. Results are mean ± sd percentage compared to control of 3 independent experiments performed in duplicate. *P < 0.05.