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. 2022 Oct 12;13:916098. doi: 10.3389/fimmu.2022.916098

Figure 6.

Figure 6

SNX17 overexpression increased LRP4 expression and MuSK phosphorylation in the gastrocnemius muscle of EAMG mice and improved AChR morphology at the NMJ and leg weakness. (A) Small-animal live imaging of EAMG mice infected with AAV2/9-ZsGreen and AAV2/9-CMV-m-SNX17-3xflag-ZsGreen for 21 days in the gastrocnemius muscle of the right leg. (B) Western blotting and qPCR analyses of SNX17 protein (n = 3/group) and mRNA (n = 9/group) in the gastrocnemius muscle tissue. (C) Western blotting analyses of LRP4, MuSK, and p-MuSK expression in the gastrocnemius muscle tissue. n = 3/group. (D) AChR (red) at the NMJ of the gastrocnemius filaments was labeled with R-BTX and the area of AChRs was quantified with ImageJ. n = 6/group; Scale bar = 50 μm. (E) Hematoxylin and eosin-stained pathological analyses of cross-sections of the gastrocnemius muscle fibers of mice in each group. Black arrows indicate muscle fiber atrophy, rupture, or inflammatory cell infiltration. Scale bar = 200 μm. (F) EMG of the gastrocnemius muscle was assessed using low-frequency RNS. The attenuation rate of the fifth amplitude of EMG compared with the first amplitude was calculated. SNX17 OE-con: n = 3; SNX17 OE: n = 5. (G) Bar dynamometer measurement of the right leg strength of mice in each group. NC: n = 6; SC: n = 5; SNX17 KD-con: n = 5; SNX17 KD: n = 8. All data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ###P<0.001. AChR, acetylcholine receptor; EAMG, experimental autoimmune myasthenia gravis; LRP4, low-density lipoprotein receptor-related protein 4; MuSK, muscle-specific receptor tyrosine kinase; NMJ, neuromuscular junction; R-BTX, rhodamine-labeled bungarotoxin; RNS, repetitive nerve stimulation; SC, solvent control; SEM, Standard Error of Mean; SNX17, sorting nexin 17.