Fig. 6

Neurotrophic factors are induced by FTY-P via S1P receptor types 1 and 3. a Human U373 astrocytoma cells were treated with FTY-P (1 μM) or S1P-receptor 1 (S1PR1) specific (SEW-2871, 1 and 10 μM), or S1P-receptor 3 (S1PR3) specific (CYM 5541, 1 and 10 μM), agonists and then left untreated (left panel) or stimulated with TNF (0.025 μg/ml) 1 h later (right panel). Supernatants were harvested 16 h later, and LIF production was detected by ELISA; mean ± SEM of three independent biological replicates. b Human U373 astrocytoma cells were pretreated with S1PR1 specific (W146, 1 and 10 μM), or S1PR3 specific (TY52156, 1 and 10 μM) antagonists, stimulated with FTY-P (1 μM), and then left untreated (left panel) or stimulated with TNF (0.025 μg/ml) 1 h later (right panel). Supernatants were harvested 16 h later, and LIF production was detected by ELISA; mean ± SEM of three independent biological replicates. c, d Human U373 astrocytoma cells were transfected with a control siRNA or two different siRNAs (2 nM) targeting S1PR1 and S1PR3, respectively, using Lipofectamine RNAimax. c Knock-down was validated by quantitative PCR; mean ± SEM of four independent biological replicates. d Twenty-four hours after siRNA transfection, cells were stimulated with FTY-P (1 μM). Eight hours later, supernatants were harvested, and LIF (left panel) and IL11 (right panel) production was determined by ELISA; representative experiment of four independent biological replicates