S1P receptor 1 (S1PR1) in FTY720-induced cell death. (A) FTY720 but not S1P induced ERK1/2 dephosphorylation in CLL B cells. CD19⁺ B cells (10⁷) from patients with CLL were incubated with DMSO, FTY720 (5 μM), or S1P (1 μM) for 3 hours, and cell lysates were subjected to Western blotting as described in “Western blotting.” (B) S1P failed to prevent FTY720-induced cell death. CD19⁺ B cells (2 × 10⁶) from patients with CLL were pretreated with indicated concentrations of S1P for 2 hours, followed by DMSO or FTY720 for 24 hours. The cells were stained with annexin V–FITC and PI as described in “Analysis of cell viability and apoptosis.” The cells were analyzed by flow cytometry, and data were collected under list mode. The data shown represent the percentage of annexin V⁻/PI⁻ viable cells plus or minus SD that are normalized to media control (n = 3). (C) Expression of S1P₁ in primary CLL B cells and B-cell lines. CD19⁺ B cells (10⁷) from patients with CLL or Raji, Ramos, MEC-1, and MEC-2 B cells were lysed and subjected to Western blotting as described in “Western blotting.” Cell lysates from mouse brains were used as positive control (representative data from 2 experiments).