SK1-I and siSphK1 decrease cellular proliferation and viability. (A) U937 cells or (B) Jurkat cells (10⁵ cells/mL) were cultured in medium containing 10% serum in the absence or presence of the indicated concentrations of SK1-I. Cell numbers were determined with a Coulter counter model Z1 (Beckman Coulter). (C) Knockdown of SphK1 by siRNA reduces cell growth. U937 cells were transiently transfected siRNA targeted to SphK1 (○) or siRNA control (●). Cells (10⁵ cells/mL) were then cultured in medium containing 10% (top panel) or 2% (middle panel) serum for the indicated times and cell numbers determined with a Coulter counter. (Bottom panel) Equal amounts of cell lysate proteins (60 μg) from duplicate cultures were separated by SDS-PAGE and immunoblotted with anti-SphK1 antibody. Blots were stripped and probed with actin antibody to ensure equal loading and transfer. SphK1 mRNA levels were normalized to actin and the ratio relative to siControl from 3 independent experiments is shown. *P < .05. (D-G) SK1-I promotes apoptosis. U937 cells cultured in 10% serum were treated without or with 10, 15, or 20 μM SK1-I for the indicated times (D) or with 20 μM SK1-I for 24 hours (E) and stained with annexin V/PI, and apoptosis was determined by flow cytometry. Early apoptotic cells are annexin V positive, late apoptotic cells are both annexin V and PI positive, whereas necrotic cells are PI positive only. (F) Duplicate cultures were treated with the indicated concentration of SK1-I for 24 hours, and the fraction of TUNEL-positive cells was determined. (G) U937 cells were cultured in medium containing 2% serum in the absence or presence of 10 μM SK1-I for 24 hours and then stained with annexin V/PI, and apoptosis was determined by flow cytometry. The percentages in the lower right quadrant correspond to early apoptotic cells (annexin V positive), whereas percentages in the upper right quadrant correspond to late apoptotic cells (annexin V/PI-positive).