Extended Data Fig. 15: Nuclear export of NMNAT1 in neurons.

a, Representative live-cell images of NMNAT1 upon treatment with TRAM 1 or unlinked warheads 2 and 3 at 10 nM for 24 h. b, Representative images of before and after axotomy on explants. Axotomies were performed using a biopsy punch, the mRuby3-Axontag protein serves as an axonal marker, Axontag = GAP43at-mRuby3-ecDHFR. c, Representative images of axons and axon termini post-axotomy, that have been pre-treated with unlinked controls for 24 h prior. d, Representative images of mNMNAT1 in axons after exposure to 1 for 24 h followed by axotomy. mRuby3 signal was used to monitor degeneration across treated neurons. d, Quantification of axon degeneration over time. e, Quantification of axon degeneration over time in axon termini. f, Comparison of the degeneration index in axona at different time points with different treatment conditions. g, Comparison of the degeneration index (DI) in the axon termini at different time points with different treatment conditions. DI was calculated as the ratio of fragmented axon area over total axon area. For plots in d, f, a total of 12 replicates derived from 4 different embryos were combined in the plots for all conditions except 1 nM 2 + 1 nM 3. A total of 6 replicates derived from 2 different embryos were combined in the plots for the 1 nM 2 + 1 nM 3 condition. For plots in e, g, a total of 6 replicates derived from 4 different embryos were combined in the plots for all conditions except 1 nM 2 + 1 nM 3. A total of 4 replicates derived from 2 different embryos were combined in the plots for the 1 nM 2 + 1 nM 3 condition. Data in d, e is shown as the mean ± s.e.m. values. Data in f, g, is shown as the mean ± s.d. values. P values in d, e, were determined by two-way ANOVA with Tukey’s post hoc test comparing the 1 nM treatment of 1 with the DMSO (purple) or the unlinked warhead control (green) at each time point. P values in f, g, were determined by one-way ANOVA with Dunnett’s post hoc test comparing each treatment to the DMSO control at each time point. Images in a, b, c are representative of neurons harvested from 4 different embryos. Scale bars are 20 µm.