SB203580 does not prevent ceramide-induced apoptosis. MC/9 cells growing in IL-3 were treated with SB203580 (1 μmol/L; •) or vehicle (▪) for 15 minutes, and then treated with C2-ceramide (50 μmol/L; ○ and □, respectively). At the indicated times, cells were removed and the presence of phosphatidylserine on the cell surface was quantitated by annexin-V-FITC staining as described in the Methods. Cells staining positive for PI were not included in the analysis. The percentage of annexin-V-FITC–positive/PI-negative staining cells never surpassed 35%, although total cell death (annexin-V-FITC– and PI-positive cells) steadily climbed and reached about 60% by 9 hours in the C2-ceramide or C2-ceramide plus SB203580 treated cells. Representative experiment of 3 is shown, with each point being the mean of duplicate determinations, and with error bars representing the range.