Extended Data Fig. 10: Efficiency of SNX25 deletion in Snx25Cx3cr1-cKOAi32Tg/+ mice.
(a) Scheme depicting dermal injection of 4-OHT into the right hind paw and of vehicle into the left hind paws of a Snx25Cx3cr1-cKOAi32Tg/+ mouse and experimental schedule. (b) Confocal microscopic images of the hind paw skin immunolabeled for YFP and SNX25 in Snx25Cx3cr1-cKOAi32Tg/+ mice that received 4-OHT injection daily for 7 days. Arrows denote SNX25- cells and arrowheads denote SNX25+ cells in YFP-expressing cells. Scale bar, 50 μm. (c) Proportion of SNX25- cells (green column) in YFP+ cells. Note that 96.0% of YFP+ cells were SNX25-. Numbers inside columns were the actual numbers of cells counted. n = 30 hind paw skin sections from 3 different mice. (d) Scheme depicting injection of 4-OHT into surgically-exposed L4 DRG of Snx25Cx3cr1-cKOAi32Tg/+ mice and experimental schedule. (e) Confocal microscopic images of DRG double labeled for YFP and SNX25 in the mice indicated in d. Scale bar, 50 μm. The summary graph shows the proportion of SNX25- cells (green column) in YFP+ cells. Note that 91.2% of YFP+ cells were SNX25-. Numbers inside columns are the actual numbers of cells counted. n = 16 sections from 3 different mice. (f) Schematic representation of how SNX25 in dMacs set pain sensitivity via Nrf2–NGF/TrkA signaling and of how Snx25 cKO resulted in a dull phenotype. Both Snx25+/− and Snx25Cx3cr1-cKO mice display reduced pain responses under both normal and painful conditions. SNX25 inhibits the ubiquitination and subsequent proteasome degradation of Nrf2 and thereby maintains NGF production and secretion into tissues. Snx25 cKO, in turn, accelerates Nrf2 degradation and lowers NGF levels in dermis, which results in a dull phenotype. Representative of three independent experiments (b and e).
