Fig. 2.

Effects of NF-κB on IL-1β–induced Wnt5a expression and neuronal differentiation. a NPCs were treated with IL-1β (10 ng/ml) for the indicated time durations, lysed, and harvested. Western blotting was performed using anti-p-p65 NF-κB, anti-p65 NF-κB, or anti-calnexin antibodies to detect the respective protein bands. b-d Cells were transiently transfected with control siRNA or NF-κB p65 siRNA for 48 h, and then treated for 2 h (b and c) or 6 h (d) with IL-1β (10 ng/ml). mRNA levels of Wnt5a were estimated by RT-PCR (b) and real-time RT-PCR (c). n = 3. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with control siRNA/IL-1β. d Western blotting was performed using anti-p65 NF-κB, anti-Wnt5a, or anti-calnexin antibodies to detect the respective protein bands. e and f Cells were transiently transfected with control siRNA or NF-κB p65 siRNA for 48 h, and then treated for 6 h with IL-1β (10 ng/ml). mRNA levels of Nt3 and Ngn1 were analyzed by RT-PCR (e) and real-time RT-PCR (f). n = 3. Data are mean ± SD; Student’s t test. * p < 0.05, ** p < 0.01 compared with control siRNA/IL-1β. g Cells were transiently transfected with control siRNA or NF-κB p65 siRNA for 48 h, and then treated for 2 days with IL-1β (10 ng/ml). Western blotting was performed using anti-p65 NF-κB, anti-NT3, anti-Ngn1, 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. h and i Cells were transiently transfected with control siRNA or NF-κB p65 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. i 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 as compared to control siRNA/IL-1β