Fig. 1. Mass spectrometry of i³Neuron axons reveals a vincristine-induced upregulation of mitochondrial proteins.

A Schematic representation of i³Neuron differentiation (created using Biorender). B Representative image of i³Neuron cell bodies plated in the center of an 8-well slide to allow axons to grow outwards. Immunostaining for βIII tubulin (orange), DAPI (blue). 20X magnification (Scale bar = 100 µm). C Representative images of axon degeneration upon addition of 5 nM vincristine for 0 (UT), 4, 8, or 24 h. Immunostaining for βIII tubulin (orange), DAPI (blue) (Scale bar = 5 µm). D Axon degeneration index (ADI) quantification for i³Neuron-treated axons with 5 nM vincristine. N = 5 independent differentiations. One-way ANOVA, Bonferroni correction (p < 0.05 *, p < 0.01 **). E Schematic representation of Boyden chambers for axonal separation and experimental procedure (created using Biorender). F Western blotting showing efficient isolation of axons from cell bodies. The nuclear envelope protein Lamin A is absent from the axon fraction. G Volcano plot showing the significant proteins up (red) and down (blue) regulated in i³Neuron axons after 5 nM vincristine treatment for 4 h (>2 unique peptides detected, fold change (FC) > 1.5, adjusted P-value < 0.05.). H Dot plot showing top 5 enriched cellular compartment gene ontology analysis categories for axon-enriched proteins after 5 nM vincristine treatment ranked by gene ratio. I Dot plot showing top 5 enriched biological process gene ontology analysis categories for axon-enriched proteins after 5 nM vincristine treatment ranked by gene ratio. J Protein–protein interaction network for proteins upregulated in the axon after vincristine treatment. Mitochondrial proteins are highlighted in red and electron transport chain proteins in blue. Proteins involved in the catalysis of ROS were also elevated (Superoxide dismutase 2 (SOD2), Thioredoxin-dependent peroxide reductase (PRDX3), Glutaredoxin-1 (GLRX) and Thioredoxin Domain Containing 17 (TXNDC17)).