Fig. 7: Analysis of mitochondria in E18 cortical neurons from CMT2B mutant mice.

E18 cortical neurons from + /+, fl/+ fl/fl embryos were dissected, cultured and assayed for mitochondrial MMP, motility as in Fig. 6. a–c: Representative images of mitochondria in neurites of E18 cortical neurons of + /+, fl/+ and fl/fl. d: Ratiometric measurements of MMP. In e, Representative images of mitochondrial morphology in cortical neurons of + /+, fl/+, fl/fl. f: Aspect ratio measurements. The numbers of mitochondria were analyzed are: n = 170 for + /+, n = 125 for fl/+, n = 121 for fl/fl. Mitochondrial transport in neurites of cortical neurons of + /+, fl/+, fl/fl was also captured by time lapsed imaging. The image series were used to generate kymographs and a representative image for each genotype is shown in g. White arrows indicate trafficking in anterograde direction, yellow arrows indicate trafficking in retrograde direction. Average velocities of axonal mitochondria moving in the anterograde (The numbers of mitochondria were analyzed are: n = 84 for + /+, n = 59 for fl/+, n = 65 for fl/fl) (h) and retrograde (The numbers of mitochondria were analyzed are: n = 74 for + /+, n = 65 for fl/+, n = 59 for fl/fl) (i) direction. j: % of retrogradely moving mitochondria. The numbers of axons in j were analyzed are: n = 20 for + /+, n = 14 for fl/+, n = 15 for fl/fl. k: % of total moving mitochondria. The numbers of axons in k were analyzed are: n = 20 for + /+, n = 14 for fl/+, n = 16 for fl/fl. Results are shown as mean ± SEM. Significance analysis was carried out using Prism. Statistical significances were calculated by One-Way ANOVA. n.s. = non significance. All p values are shown in the graphs. In l, 2 month-old brain lysates from + /+ (n = 6), fl/+ (n = 6), fl/fl (n = 6) were generated and 20 μg proteins were analyzed by SDS-PAGE and immunoblotting with indicated antibodies. The relative levels of Drp1 (m) and TOM20 (n) were quantitated and normalized against β-Actin using BioRad-Image Lab. Results are shown as mean ± SEM. Significance analysis was carried out using Prism. Statistical significances were calculated by One-Way ANOVA. n.s. = non significance. All p values are shown in the graphs.