Fig. 4: Protection of neurites from AIND by pharmacological inhibition of SARM1.

2.5D LUHMES were treated with different 5-iodoisoquinoline (5-IIQ) concentrations 30 min before axotomy. Neurites were analyzed 18 h after axotomy. A Neurites were stained with calcein-AM and TMRE and imaged by epifluorescence microscopy 18 h after axotomy. Representative images of calcein stained neurites are shown. Scale bar = 200 µm. B Neurite fragmentation and integrity were quantified by an image analysis algorithm at 18 h after cut. For each biological replicate, 10 fields (each covering 0.32 mm²) of 3-5 technical replicates of calcein stained neurites were recorded. Data is from three independent biological replicates. C The number of TMRE⁺ mitochondria was determined by an image quantification algorithm. Images were recorded as in (B). Data for each image were normalized to the neurite area in the image. In the end, data were normalized to uncut controls. **p < 0.01, ***p < 0.001 by ANOVA with Dunnet’s post hoc test. D ATP was measured after lysis of all neurites in a well. Neurites were treated with or without 50 µM 5-IIQ 30 min before axotomy. Data was normalized to ATP measured in freshly isolated neurites (t = 0 h). “Ctrl” refers to untreated neurites 18 h after axotomy. **p < 0.01 by Student’s t test. E Isolated neurites were obtained as above, but they were grown on glass coverslips with minimal Matrigel coating. Samples were fixed and processed before imaging by scanning electron microscopy (SEM). Representative images are shown. Scale bar = 10 µm.