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. 2023 Jan 26;24(3):439–451. doi: 10.1038/s41590-022-01418-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 3 — (a) VF thresholds of young (2 and 3 weeks after birth) WT (n = 8) and Snx25^+/− mice (n = 6). 2w, p = 0.288; 3w, p = 0.016. g, gram. (b) Size distribution of DRG neuron diameters is plotted for 3-week-old WT and Snx25^+/− mice (L4 level, WT (blue columns): 1551 cells from 3 mice; Snx25^+/− mice (red columns): 2036 cells from 3 mice). X-axis values indicate the maximum diameter in each 5-μm range. Numbers above or inside columns are the actual numbers of cells in each diameter range. (c) Cumulative frequency distribution of DRG neuron diameters. (d) Representative confocal microscopic images showing CGRP- and NF200-immunoreactive neurons in the DRG (L4) of WT and Snx25^+/− mice. Scale bars, 100 μm. (e) Left, percentage of CGRP⁺ cells among Nissl-positive cells (WT: n = 8; Snx25^+/−: n = 9 DRG sections from at least 3 different mice of each genotype, p = 0.27). Right, percentage of NF200⁺ cells among Nissl-positive cells (WT: n = 8; Snx25^+/−: n = 9 DRG sections from at least 3 different mice of each genotype, p = 0.544). (f) Representative images of PGP9.5⁺ immunoreactivities in the plantar skin of WT and Snx25^+/− mice. Arrowheads indicate neuronal fibers labeled for PGP9.5. Scale bar, 50 μm. (g) PGP9.5⁺ region per unit area (μm²) was measured and plotted for each genotype (WT: 44 sections from 3 mice; Snx25^+/−: 68 sections from 4 mice, p = 0.805). (h) Representative immunoblot showing TRPV1, TrkA, and SNX25 proteins in the sciatic nerve and spinal cord of WT and Snx25^+/− mice. (i) Representative immunofluorescence images showing TrkA-immunoreactive terminals (green fluorescence, arrowheads) in the dorsal horn of the WT and Snx25^+/− mice counterstained with DAPI (blue fluorescence). Right, quantification of mean fluorescence intensity (WT: n = 5; Snx25^+/−: n = 6 spinal cord sections from 3 different mice of each genotype, p = 0.066). Scale bar, 100 μm. Results are represented as mean ± SEM. Statistical analyses were performed using the two-tailed Student’s t-test. *p < 0.05. n.s., not significant. Representative of three independent experiments (d, f, and i).

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