Sphk2 expression in apoptotic cells contributes to the release of S1P. (A) Apoptosis in human primary macrophages was induced for 8 hours with 10 ng/mL TNF-α and 10 μg/mL CHX. Cells remained untreated or were treated with ACM or ACM from Jurkat cells nucleofected with siRNA to target Sphk1 or Sphk2, 16 hours prior to induction of cell death. Caspase-3 activity was quantified as described in “Materials and methods.” Data are presented as the mean ± SD from 4 independent experiments. Significant differences between samples are marked with an asterisk (P < .05). (B) Jurkat cells were nucleofected with control siRNA or siRNA against Sphk1 or Sphk2. Western analysis shows Sphk1 and Sphk2 expression. One representative experiment of 3 is displayed.