Fig. 4

CHD8 regulates neurite growth in vitro. a shRNA-mediated knockdown of mouse Chd8 in transfected cultured neurons. Upper, representative western blots; lower, quantification of western blot analysis. shRNA#1, 0.14 ± 0.095; shRNA#2, 0.60 ± 0.14; shRNA#1, 0.77 ± 0.089. For ANOVA, P = 0.587. For post hoc Dunnets’ test, **P = 0.004 for shRNA#1 vs shRNA-scr; *P = 0.041 for shRNA#2 vs shRNA-scr; *P = 0.046 for shRNA#3 vs shRNA-scr. All data were shown in mean ± SEM and were collected from three to six independent experiments. b Immunostaining showing shRNA-mediated knockdown of Chd8 in transfected neurons in mouse brains. Immunohistochemical staining of endogenous CHD8 in coronal sections from a P0 mouse brain electroporated with shRNA#1 and GFP at E14.5. The open arrows indicate successfully transfected cells, and the filled arrows indicate normal cells. Scale bar, 10 μm. c Representative images of DIV3 cultured neurons transfected with GFP and the plasmids indicated. Constructs were transfected at DIV0, cultured for 3 days in vitro, and then immunolabeled with anti-GFP. Scale bar, 100 μm. d Quantification of the total axon length of neurons transfected with Chd8 shRNA constructs. n = 126, 133, 124 neurons per group, respectively. Total axon length: shRNA-scr, 196.0 ± 8.42 μm; shRNA#1, 160.4 ± 7.56 μm. For ANOVA, P = 0.01. For post hoc Dunnets’ test, **P = 0.0032 for shRNA#1 vs shRNA-scr; shRNA#2, 168.8 ± 6.98, **P = 0.0052 for shRNA#2 vs shRNA-scr. e Defective axon growth in Chd8 shRNA#2 construct was rescued in neurons by co-transfected with shRNA#2-resistant hCHD8 constructs (n = 50 neurons per group). Total axon length: shRNA-scr, 202.6 ± 9.13 μm; shRNA#2, 168.2 ± 6.68 μm. For ANOVA, P = 0.07. For post hoc Dunnets’ test, **P = 0.004 for shRNA#2 vs shRNA-scr; shRNA#2 + hCHD8, 214.4 ± 6.15, P = 0.32 for shRNA#2 + hCHD8 vs shRNA-scr. f Representative images of DIV7 cultured neurons transfected with GFP and Chd8 shRNA constructs as indicated. g Quantification of dendritic length in neurons transfected with Chd8 shRNA constructs as indicated. n = 80–100 neurons per group. Mean dendritic length: shRNA-scr, 130.1 ± 6.3 μm; shRNA#1, 90.7 ± 4.61 μm. For ANOVA, P < 0.001. For post hoc Dunnets’ test, ***P < 0.0001 for shRNA#1 vs shRNA-scr; shRNA#2, 104.5 ± 9.53, *P = 0.032 for shRNA#2 vs shRNA-scr. Total dendritic length: shRNA-scr, 694.9 ± 42.5 μm; shRNA#1 is 435.9 ± 30.6 μm. For ANOVA, P = 0.0007. For post hoc Dunnets’ test, ***P = 0.0001 for shRNA#1 vs shRNA-scr; shRNA#2 is 425.3 ± 39.2 μm, ***P = 0.0003 for shRNA#2 vs shRNA-scr. h Quantification of dendritic complexity as measured by Sholl analysis (n = 50–80 neurons per group). For ANOVA, P = 0.0015. For Bonferroni post-tests, *P < 0.05; **P < 0.01; **P < 0.001. i Co-expression of the human CHD8 rescued the defective dendritic growth by the shRNA-#2 in DIV7 neurons. n = 100–120 neurons in each group. Data represent mean ± SEM. For ANOVA, P = 0.0127 (left panel); P = 0.0013 (right panel). For post hoc Dunnett’s test, *P < 0.05; **P < 0.01; ***P < 0.001. j Quantification of dendritic complexity as measured by Sholl analysis (n = 50–80 neurons per group). For ANOVA, P < 0.001. For Bonferroni post-tests, *P < 0.05; **P < 0.01; ***P < 0.001