Fig 2: The effects of Bhlhe40 over-expression on differentiation and metabolism. A Tet-off system was established to stably over-express Bhlhe40 and GFP simultaneously in C2C12 cells (C2C12-Bhlhe40), in which the expression is shut off in the presence of Doxycycline (Dox, 25 ng/ml). The expression of Bhlhe40 mRNA (left panel) and protein (right panel) in myoblasts is shown in (A) and its effects on myogenic differentiation (B) was represented by the fusion index (nuclei number in myotubes/total nuclei number). The effects on mtDNA levels of CMB and MT cells, and on Mitotracker stain intensity, SDH activity, and ROS levels in myotubes are shown in (C), (D), (E), and (F), respectively. (G) Images of C2C12-Bhlhe40 myoblasts transfected with KillerRed-PTS1 expression vector and counterstained with DAPI. The distribution (%) of cells with different numbers of peroxisome/cell and the average peroxisome number/cell are shown in (H) and (I), respectively. * and * *: p < 0.05 and p < 0.01 vs. Dox (+) cells.

Fig 3: Western blot images (A) and quantifications (B–G) of cell lysates from C2C12 myotubes with control or UCHL1 siRNA knockdown for perilipin 2 (B), perilipin 5 (C), CD36 (D), HSL (E), MAGL (F), and SDHB (G). n = 4 per group.

Fig 4: GLUD2 mutation modulates SDH expression in MPTP-treated mice and MPP+-treated U251 cells.a, b Relative intensity of fumaric acid and succinic acid in the SN of AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups. n = 6. c, d ROC analysis for fumaric acid and succinic acid. e, f SDH levels in the serum and SN of AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups. n = 5. g ATP levels in the SN of AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups. n = 6. h, i The effect of GLUD2 or its mutant on SDHA and SDHB expression in the SN of MPTP-treated mice and MPP+-treated U251 cells were determined by western blotting. n = 6 in (h) and n = 3 in (i). j Immunofluorescent staining of EGFP and SDHA in MPP+-treated U251 cells expressing WT or mutant GLUD2 (scale bars, 10 µm). Results are expressed as the mean ± SEM. **p < 0.01, *p < 0.05 vs. Control group. ##p < 0.01, #p < 0.05 vs. MPTP + AAV-GFP or MPP+ group. &&p < 0.01 vs. MPTP + AAV-GLUD2 group. Statistical significance was determined by one-way ANOVAs and Tukey tests for post-hoc comparisons.

Fig 5: Effects of the GFM1 mutations on mRNA variants and oxidative phosphorylation complex protein expression levels in the liver and kidney tissues from the index patient. (A) The representative results of Sanger sequencing using reverse transcription PCR amplification products showing GFM1 mRNA variants in the liver and kidney tissues of the control and the index patient. (B) Western blot analysis of whole cell extracts from the liver and kidney tissues from the index patient and the control. In the index patient, decreased levels of NDUFA13 and ATP5A were observed in the liver and decreased expression of ATP5A was identified in the kidney. SDHB and GAPDH were used as loading controls. GFM1, G elongation factor mitochondrial 1; NDUFA13, NADH: ubiquinone oxidoreductase subunit A13; SDHB, succinate dehydrogenase complex iron sulfur subunit B; UQCRC2, ubiquinol-cytochrome c reductase core protein 2; ATP5A, ATP synthase peripheral stalk subunit F6.