Fig. 4. Mdivi-1 delays axon degeneration via a DRP1-independent mechanism.

A Validation of DRP1 gene knockdown (KD) using qPCR in CRISPRi-i³ iPSCs. Results are represented as mean ± SEM. N = 3. Unpaired t test. (p < 0.0001 ****). B Representative images of axonal mitochondria in control and DRP1 KD i³Neurons. Axon (cytoplasmic blue fluorescent protein (BFP)) and mitochondria (mitoGFP). Scale bar = 10 µm. C Quantification of mitochondrial area in control and DRP1 KD i³Neurons. Results are represented as mean ± SEM. Over 2000 mitochondrial particles quantified from 3 independent differentiations. Unpaired T test (p < 0.001 ***). D Representative images of control and DRP1 KD i³Neuron axons for 0, 4, 8, and 24 h with 5 nM vincristine for axon degeneration quantification. Immunostaining for βIII tubulin (orange). Scale bar = 10 µm. E Axon degeneration index of the experiments shown in (D). Results are represented as mean ± SEM. N = 3 independent differentiations, 5–8 images per differentiation. Two-way ANOVA, Bonferroni correction. No significant differences. F Representative images of control and DRP1 KD i³Neuron axons UT or treated for 24 h with 5 nM vincristine and 5 nM vincristine + 50 µM mdivi-1. Scale bar = 10 µm. Axon degeneration index of the experiments shown in (G). Results are represented as mean ± SEM. N = 3 independent differentiations, 8–10 images per differentiation. Two-way ANOVA, Bonferroni correction. (p < 0.05 *, p < 0.01 **).