Fig. 4

FTY-P effects are detectable also after long-term exposure. a For experiments shown in b and c, cells were switched to serum-free medium before the experiment. Serum-free cell culture medium was then replaced daily for up to 7 days. For the last n days (orange period), it contained additional FTY-P or S1P (n is displayed on the X axis in b and c). Thus, the total duration of serum-free cell culture was equal for all conditions per experiment. b U373 astrocytoma cells were treated with FTY-P (1 μM) or S1P (0.1 μM) for the last 1 and 6 days (one experiment) or the last 1, 4 and 7 days (3 experiments). Supernatants were harvested 8–16 h after the last stimulation. IL11 and LIF were measured by ELISA. Values from the vehicle control (averages for LIF 7.6 pg/ml, IL11: 2.7 pg/ml) were subtracted from the FTY-P and S1P stimulated cells. Boxplots indicate median and first/third quartile, with whiskers extending to outliers up to 1.5 × interquartile range; one-tailed Wilcoxon rank sum test. c Human astrocytes of embryonic origin (triangles) or U373 astrocytoma cells (circles) were stimulated with FTY-P (1 μM) or S1P (0.1 or 1 μM) for the last n days. Multiple data points per time point represent independent biological replicates. One hour after the last FTY-P application, TNF (0.025 μg/ml) was added. Cell lysates were harvested 8–16 h after TNF application. BAFF mRNA, CXCL10 mRNA, and CXCL10 protein were determined by qPCR and ELISA. Values of FTY-P and S1P treated samples are displayed normalized to the samples without FTY-P and S1P (i.e. TNF only = 100 %). Boxplots indicate median and first/third quartile, with whiskers extending to outliers up to 1.5 × interquartile range; one-sample t tests