SphK2 release during apoptosis is coupled to PS exposure. Jurkat cells were killed with Sts or etoposide in the absence/presence of 100μM nifedipine or 5mM EGTA. (A) FACS dot blots display PS exposure visualized with annexin V–FITC compared with cell size, with cell shrinkage indicating cell death. Representative data of 4 experiments are shown. (B) Cells were stained with annexin V–FITC (□) or DiOC6 (■) and analyzed by flow cytometry. Data are means ± SEM from 4 independent experiments. (C) Histograms display caspase-3/7 (DEVD-AMC cleavage) or caspase-1 (YVAD-AMC cleavage) activity normalized to controls. Data are means ± SEM from 3 independent experiments. (D) Extracellular SphK2 activity is displayed. Data are means ± SEM from 4 independent experiments. (E) Western analysis of intra- and extracellular SphK2 expression. (F) Western analysis of intra- and extracellular SphK2 and intracellular caspase-1–p20 as well as caspase-3–p17 expression from the same gel. (B-D) Asterisks indicate significant differences compared with cells treated with Sts alone. *P < .05; **P < .01; ***P < .001.
