FTY720-induced toxicity in the Ramos B-cell line is dependent on activation of PP2a. (A) FTY720-induced PP2a activity in the Ramos B-cell line. Ramos B cells (105/mL media) were incubated with DMSO, 10 μM FTY720, or 100 μM 1,9 di-deoxy-forskolin for 4 hours. The PP2a activity in the cell lysates was measured as described “PP2a activity (nonradioactive assay).” The results shown are representative of 2 independent experiments. (B) FTY720-induced PP2a activity in the Ramos B-cell line is inhibited by okadaic acid. Ramos B cells (105/mL media) were pretreated with media or indicated concentrations of okadaic acid for 2 hours, followed by incubation with DMSO or 10 μM FTY720 for 4 hours. The PP2a activity in the cell lysates was measured as described in “PP2a activity (nonradioactive assay).” The results shown are representative of 2 to 3 independent experiments. (C) FTY720-induced cellular toxicity is partially rescued by okadaic acid in Ramos B cells. Ramos B cells (105/mL media) were pretreated with media or indicated concentrations of okadaic acid (5 nM) for 2 hours, followed by incubation with DMSO or 10 μM FTY720. 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 the 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. The results shown are the means (± SD) of 5 independent experiments.
