Fig. 1

Effects of IL-1β-induced Wnt5a expression on neuronal differentiation. NPCs were treated with IL-1β (10 ng/ml) for the indicated time durations and mRNA levels of Nt3, Ngn1 were analyzed by RT-PCR (a) and real-time RT-PCR (b). n = 3. Data are mean ± SD; Student’s t test. * p < 0.05, ** p < 0.01, ^††p < 0.01 compared with 0 h control, for Nt3 and Ngn1 respectively. c and d NPCs were treated with IL-1β (10 ng/ml) for 3 days, and they were stained with anti-Tuj1 to visualize neurite extensions. Scale bar, 20 μm. d 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 < 0.001 compared with untreated control. e NPCs were treated with IL-1β (10 ng/ml) for 2 h. mRNA levels of Wnt3a, Wnt5a, Wnt5b, Wnt7a, and Wnt7b were analyzed by RT-PCR (left). mRNA level of Wnt5a was analyzed by real-time RT-PCR (right). n = 3. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with control. f NPCs were treated with IL-1β (10 ng/ml) for the indicated time durations, and cells were lysed. Western blotting was performed using anti-Wnt5a or anti-calnexin antibodies to detect the respective protein bands. Graphs show mean densities as percentage change for three independent experiments (n = 3). Band intensity was quantified with Quantity Ones® software. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with 0 h control. g and h Cells were transiently transfected with control siRNA or Wnt5a siRNA for 48 h, and then treated for 6 h (g) or 2 days (h) with IL-1β (10 ng/ml). g mRNA levels of Nt3 and Ngn1 were analyzed by real-time RT-PCR. The results are based on three independent experiments (n = 3). Data are mean ± SD; Student’s t test. ** p < 0.01 compared with control siRNA/IL-1β. h Western blotting was performed using anti-NT3, anti-Ngn1, anti-Wnt5a or anti-calnexin antibodies to detect the respective protein bands (i). Cells were transiently transfected with control siRNA or Wnt5a siRNA for 48 h, and then treated for 3 days with IL-1β (10 ng/ml). They were then stained with anti-Tuj1. Scale bar, 20 μm. j Neurite lengths were measured in randomly selected fields using four independent experiments. n = 4 per group. Data are mean ± SD; Student’s t test. *** p < 0.001 compared with control siRNA/IL-1β