Fig. 5

Effects of IL-1β on Wnt5a-mediated signaling and neuronal differentiation. a NPCs were transiently transfected with control siRNA or Wnt5a siRNA for 48 h, and then incubated for 15 min with IL-1β (10 ng/ml). GTP-loaded RhoA activity was measured using a pull-down assay, as described in the Materials and Methods section. The data were normalized to the amount of total RhoA. Graphs show mean densities as fold change from three independent experiments (n = 3). Band intensity was quantified with Quantity Ones® software. Data are mean ± SD; Student’s t test. * p < 0.05 compared with control siRNA/IL-1β. b and c Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and then incubated for 6 h (B) or 2 days (c) with IL-1β (10 ng/ml). b mRNA levels of Nt3 and Ngn1 were analyzed by real-time RT-PCR. n = 3. Data are mean ± SD; Student’s t test. ** p < 0.01 as compared to control siRNA/IL-1β. c Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and then treated for 2 days with IL-1β (10 ng/ml). Western blotting was performed using anti-NT3, anti-Ngn1, anti-RhoA, or anti-calnexin antibodies to detect the respective protein bands. d and e Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and then incubated for 3 days with IL-1β (10 ng/ml). They were then stained with anti-Tuj1. Scale bar, 20 μm. e Neurite lengths were measured in randomly selected fields using three independent experiments. n = 3 per group. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with control siRNA/IL-1β. f Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and then treated for 30 min with IL-1β (10 ng/ml). Western blotting was performed using anti-p-JNK, anti-JNK, anti-RhoA or anti-calnexin antibodies to detect the respective protein bands. Graphs show mean densities as fold change for three independent experiments (n = 3). Band intensity was quantified with Quantity Ones® software. Data are mean ± SD; Student’s t test. * p < 0.05 compared with control siRNA/IL-1β. g and h Cells were pretreated with 5 μM Y27632 or 2 μM SP for 1 h, and then treated with IL-1β (10 ng/ml) for 6 h (g) or 2 days (h). g mRNA levels of Nt3 and Ngn1 were analyzed by real-time RT-PCR. n = 3. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with IL-1β-treated cells. h Cells were pretreated with 5 μM Y27632 or 2 μM SP for 1 h, and then treated with IL-1β (10 ng/ml) for 2 days. Western blotting was performed using anti-NT3, anti-Ngn1 or anti-calnexin antibodies to detect the respective protein bands. (I and J) Cells were pretreated with 5 μM Y27632 or 2 μM SP for 1 h, and then treated with IL-1β (10 ng/ml) for 3 days. They were then stained with anti-Tuj1. Scale bar, 20 μm. j Neurite lengths were measured in randomly selected fields using three independent experiments. n = 3 per group. Data are mean ± SD; Student’s t test. ** p < 0.01 as compared to IL-1β-treated cells. k Proposed model for the signaling pathway in IL-1β-mediated neurite outgrowth of cortical NPCs. The model suggests that IL-1β-induced Wnt5a plays a major stimulatory role in neuronal differentiation and that it acts through the RhoA/ROCK/JNK pathway, leading to neurite outgrowth