Fig 2: RNAi of the Ts-RNF gene affects the development and skeletal muscle autophagy level(A) The protein expression levels of Ts-RNF after siRNA interference in each group were detected by Ts-RNF Rb pAb. Fluorescently labeled siRNA interference Ts-ML was used as a negative control (NC). The mRNA expression levels of the Ts-RNF gene after siRNA interference in each group after siRNA interference. Data are shown as the means ± SEMs from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.(B) Time points for tail vein injection to induce infection in different groups and mouse diaphragm collection.(C) Confocal microscopy images of p62 in the skeletal muscle of the infected mice. p62 (red), Ts-RNF (green), and Hoechst (blue). Primary antibodies were replaced with preimmune serum, and PBS served as a control. Representative single optical sections and merged images are shown. Scale bars: 20 μm.(D) The skeletal muscle of the mouse was collected on days 3, 10, and 17 after infection with T. spiralis. Western blotting was used to detect the total protein expression and phosphorylation levels of Ts-RNF, Pink1(PTEN induced putative kinase 1), Parkin (PARK2), P62(SQSTM1), and LC3B(MAP1LC3) in the Ts group and the siRNA-Ts group. The data and images are representative of at least three independent experiments. The samples were derived from the same experiment, and the blots were processed in parallel.(E) H&E-stained sections of the mouse diaphragm on days 3, 10, and 17 after infection with T. spiralis with normal uninfected mouse skeletal muscle as a control. Scale bar: 20 μm.(F) The skeletal muscle of mouse was collected on days 3, 10, and 17 after infection with T. spiralis and the mRNA expression levels of IL-1β, TNF-α, and IL-6 in the NC group and Ts-RNF group. Data are shown as the means ± SEMs from three independent experiments (n = 3). Statistical analysis was performed using independent samples t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.

Fig 3: SkQ1 regulated PINK1/PRKN-mediated mitophagy. (A–F) LHON-IFBs were pretreated with SkQ1 (20 nM) and DMSO for 7 days, followed by treatment with 200 μM H2O2 for 6 h. The expressions of PINK1, PRKN, LC3B, SOD2, VDAC1, and β-actin protein were detected by Western blot. β-actin was used as a reference, n ≥ 4. (G,H) The expression levels of PINK1 and LC3B in HEK293T and RPE-1 cells were consistent with those of LHON-IFBs, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n ≥ 3. (I) SkQ1 could be involved in regulating oxidative stress and PINK1/PRKN-mediated mitophagy pathway.