Release of SphK2 depends on activation of caspase-1. (A) Western blots show expression of SphK2, caspase-3, and active caspase-1 (p20) in the cytosol and of SphK2 in the supernatants of Jurkat cells incubated with Sts for the times indicated. (B) Western analysis of pro–IL-1β and mature IL-1β. Jurkat cells were controls or transfected with the pro–IL1β expression vector pCACGS-p-IL-1B and treated with Sts for the times indicated. (C) Western analysis of SphK2 expression in the cytosol and supernatants of Jurkat cells. Cells were controls, treated with Sts, or Sts and 1μM caspase-1 inhibitor Ac-YVAD-CMK (C1-I). (D) THP-1 macrophages were pretreated with supernatants of apoptotic Jurkat cells (ACM), or respective supernatants generated with either DMS (ACM-DMS) or Ac-YVAD-CMK (ACM-C1-I) present. Apoptosis was induced for 8 hours with 250μM etoposide. Caspase-3 activity in THP-1 macrophages normalized to untreated cells is shown. Data are means ± SEM from 4 independent experiments. (E-F) Jurkat cells were controls or transfected with either siCONTROL or siRNA directed against caspase-1 before induction of apoptosis with Sts. (E) Western analysis shows expression of pro–caspase-1 and the caspase p20 subunit in the cytosol and t-SphK2 in the cell supernatant. (F) Histograms display caspase-3/7 (DEVD-AMC cleavage) or caspase-1 (YVAD-AMC cleavage) activity normalized to controls. Data are means ± SEM from 4 independent experiments. (G) THP-1 macrophages were pretreated with supernatants of apoptotic Jurkat cells transfected with siCONTROL (ACM-siControl) or siRNA targeting caspase-1 (ACM-siCasp1). Apoptosis was induced for 8 hours with 250μM etoposide. Caspase-3 activity in THP-1 macrophages was normalized to untreated cells. Data are means ± SEM from 5 independent experiments (*P < .05; **P < .01; ***P < .001).