Figure 4. Aberrant expression of VGF compromises neurogenesis in CIC-deficient brains.

(A) Biological replicates of CIC ChIP-seq tracks at Vgf promoter regions. (B) WB analysis in P0.5 brains of Cic^WT and Cic^KO mouse. SE, short exposure; LE, long exposure. The blots were run on same gel. Mean ± SEM of 3 experimental animals. (C) IF analysis for VGF in the P14 brains of Cic^WT and Cic^KO. Scale bar: 100 μm. (D) WB analysis for VGF in NT^KD NPC and Cic^KD NPC during differentiation. The blots were run on same gel. (E) IF analysis for Flag, VGF, NeuN, and BrdU in VGF-Flag–expressed NPC (VGF-F) and control NPC (con) at 7 days of differentiation. Scale bar: 50 μm. (F) Quantitations for NeuN⁺ or BrdU⁺ numbers are plotted. Mean ± SEM of 100 DAPI⁺ nuclei from 3 experiments. (G) WB analysis for indicated proteins in control NPC and VGF-F NPC at 5 days of differentiation. (H) WB analysis for indicated proteins in NT^KD NPC and Cic^KD NPC at 4 days of differentiation. sg-NT, nontargeted control single-guide RNA. For G and H, samples were run on 2 gels, and the representative ACTB blot is shown. (I) IF analysis for NEUN in NT^KD NPC and Cic^KD NPC at 7 days of differentiation. (J) Quantifications for images from I are plotted. Mean ± SEM of 100 DAPI⁺ nuclei from 3 experiments. Statistical significance was determined by unpaired t test for B and F and 1-way ANOVA for J. *** P < 0.001. For C, D, E, G, H, and I, 3 experiments were conducted, and each representative result is shown.