Figure 2. Netrin-1 rescue of axon initiation defect caused by Myo X deficiency. A–C, E18.5 cerebral cortices that were electroporated with GFP (Control) or Cre-GFP (Netrin-1-KO) plasmids into NTN1f/f embryos at E14.5. A, Representative images. Scale bar, 100 μm. B, Quantification of axon intensity ratio. Student's t test, p = 0.0418. C, Quantification of GFP+ cells in CP. Student's t test, p = 0.39. D, Representative images of E18.5 cerebral cortex electroporated with control miRNA (Control), Myo X miRNA (Myo X KD), or Myo X miRNA together with Myc-Netrin-1 plasmids (Myo X KD + Netrin-1) at E14.5. Scale bar, 100 μm. E, Quantification of axon intensity ratio. One-way ANOVA: p = 0.0303 for the MyoX KD group; p = 0.74 for Myo X KD + Netrin-1 group. F, G, Immunoblotting analysis of Netrin-1-KD and DCC-KD efficiency. F, HEK293T cells were transfected with a scrambled or Netrin-1 shRNA together with Myc-Netrin-1 plasmids for 48 h. Cell lysates were extracted for Myc immunoblotting. Student's t test was used for the statistical analysis of Netrin-1-KD efficiency, p = 0.0012. G, NLT cells were transfected with a control or DCC miRNA for 48 h. Cell lysates were extracted for DCC immunoblotting. Student's t test was used for the statistical analysis of Netrin-1-KD efficiency, p < 0.0001. H, Representative images of P14 cerebral cortex electroporated with Control miRNA (Control), Netrin-1 shRNA (Netrin-1 KD), and DCC miRNA (DCC KD) at E15.5. Scale bar, 500 μm. I, Quantification of axon contralateral branching. Student's t test, p = 0.39. J, Quantification of axon contralateral branching by using the cortical/midline intensity ratio, which is defined as axonal GFP intensity (marked by a white rectangle) over the midline GFP intensity (marked as a red rectangle), as illustrated in the schematic diagram. Student's t test, p < 0.0001. Data are presented as the mean ± SEM. The numbers of brain sections scored are from three different brains for each group and indicated on the graphs. ns, No significant difference; *p < 0.05; **p < 0.0; ***p < 0.001.