Fig 2: Western blot of muscle tissue. (A–C) Representative blots used in the quantification of protein expression of muscle tissue from five patients with compound heterozygous SPG7 mutations and three control subjects. Testing was performed in triplicate and quantification of aggregate mean with SD (normalized to GAPDH) are represented in D. Markers of mitochondrial mass, including SDHA, porin, and HSP60 are significantly increased in SPG7 patients compared with controls. SPG7 and OPA1 were also significantly elevated in SPG7 patients compared with controls. AGF3L2 was decreased compared with controls. All the above were statistically significant to *P < 0.01.

Fig 3: Representative western blot and densitometric analysis showing level of PGC1a (a), MFN1 (b), MFN2 (c), OPA1 long form (L-OPA1, 95kD) and short form (S-OPA1, 75kD) (d), and DRP1 (e) proteins in RMC cells following 0, 30, and 60 µM of bicalutamide treatment for 24 and 48 h. ß-Actin is as cytosolic normalization control; HSP60 is as mitochondrial normalization control. Bar graph represents the mean ± SD of triplicate experiments: * p < 0.05, ** p < 0.01 *** p < 0.001 compared to Bic 0 µM at 24 h. ## p < 0.01 compared to Bic 30 µM at 24 h. § p < 0.05, §§ p< 0.01, §§§ p < 0.001 compared to Bic 0 µM at 48 h. p < 0.001 compared to Bic 30 µM at 48 h.

Fig 4: (a) Intracellular NAD+ level in RMC cells treated with 0, 30, and 60 µM of bicalutamide at 3, 24, and 48 h. (b) Representative western blot and densitometric analysis showing level of SOD2 protein in RMC cells following 0~60 µM of bicalutamide treatment for 24 and 48 h (HSP60 is shown as normalization control). Triplicate experiments were performed and expressed as mean ± SD. *** p < 0.001 compared to the normal control. ### p < 0.001 compared to Bic 30 µM. p < 0.05, §§§ p < 0.001 compared to Bic 30 µM at 48 h.

Fig 5: A3A and A3B were contained in the mitochondria. (a) W12 cells were transfected with an expression vector coding A3A or HA-A3B, and cultivated in maintenance medium for 48 h. Their lysates were fractionated into cytosolic and mitochondrial fractions, and subjected to western blotting analysis. (b,c) W12 cells were induced to differentiate under high extracellular calcium concentration, and fractionated into cytosol and mitochondrial fraction, (b) Each fraction was subjected to immunoblotting of A3s, HSP60 (a mitochondrial marker), and ß-tubulin. (c) DNA was extracted from each fraction and subjected to 3D-PCR analysis targeting COI.