Fig. 5

Axonal development requires mtIF3-dependent mitochondrial translation in growing axons. a Timeline for the experiment. Primary hippocampal neurons were cultured on a microfluidic device. At DIV4, chloramphenicol (CA) was added to either cell body or axonal chamber. After 30 min, BDNF was added to the axonal chamber. Neurons were stained and imaged at DIV5. b Representative images of axons. Axons were marked by Tau-1 immunostaining (scale bar, 200 μm). c The axonal length was measured from the exit border of microgrooves (dotted lines), including the main axons and branches. Data represent mean ± SEM (N = 4–5 replicates and n = 422–745 axons). n.s., not significant; ****P < 0.0001, as determined by Aligned ranks transformation ANOVA with Wilcoxon rank-sum test. d Representative images of primary hippocampal neurons expressing shRNA and mtIF3 (SR). Hippocampal neurons were co-transfected with shRNAs and overexpression vectors and then treated with BDNF at DIV1. The axonal length was measured at DIV3 (scale bar, 100 μm). TagRFP657 and mito-mTFP1 signals confirm co-transfection of shRNAs and overexpression vectors. White arrow head indicates the start point of axon and blue arrow head indicates the end point of axon. e Axon length was calculated by measuring TagRFP657 signals. Data represent mean ± SEM (N = 5 replicates and n = 50 neurons). Aligned ranks transformation ANOVA detected significant interactions of mtIF3 (SR) overexpression and BDNF on the axon length in mtIF3 shRNA groups (but not in control shRNA groups) (P = 0.0002). n.s., not significant; *P < 0.05, ****P < 0.0001, as determined by Wilcoxon rank-sum test