FIGURE 4

TFG deficiency impairs neurite outgrowth and causes neuronal apoptosis. (A and B) The efficiency of the small interfering RNA (siRNA) to suppress endogenous TFG expression was demonstrated by western blot. (mean ± SD; two‐tailed unpaired t‐test, *p  < 0.05; n =  3). (C) Immunofluorescence staining of primary cultured neurons on day 8 after Tfg knockdown. Neurites were labeled with MAP2 (green), and the nucleus with DAPI (blue). Scale bars: 50 μm. (D) Immunofluorescence staining of primary cultured neurons on day 17 after Tfg knockdown. Neurons were transduced with lentivirus with mCherry (red), neurites were labeled with MAP2 (green) and the nucleus was labeled with DAPI (blue). Scale bars: 20 μm. (E) TUNEL staining of control and Tfg knocked‐down neurons on day 17 in culture. (F) Gross morphology of zebrafish (hb9:eGFP) embryos at 2 dpf. Blue dotted lines show the curved body axis in both groups of tfg morphants. Dpf, days postfertilization. (G) Quantification of the average motor axon length. (mean ± SEM; ANOVA, ***p < 0.0001; n = 10). (H) Acridine orange staining of zebrafish at 32 hpf. Apoptotic cells are visible as bright green spots or black spots. The red dotted box shows the brain and spinal cord region of zebrafish. The boxed regions are shown at higher magnification on the bottom. (I) Quantification of apoptosis particle number in the brain and spinal cord (mean ± SEM; ANOVA, ***p < 0.0001; n = 10).