Fig 2: Expression levels of SMC contractile genes are altered with knockdown of COQ8B(A) RNA levels of COQ8B and genes important for SMC contraction measured by qRT-PCR 48 h after siRNA knockdown of COQ8B (siCOQ8B) or negative control siRNA (siNeg). Each point represents a sample extracted from an independently transfected well. Target gene RNA levels are adjusted for ACTB and displayed relative to mean of siNeg SMCs. Shaded bars show means ± standard error. Levels were compared between conditions using Student’s t test. *p < 0.05; **p < 0.01.(B) Western blot of calponin-1 (CNN1) levels between siCOQ8B and siNeg SMCs in protein extracted concurrently with RNA samples. Ratios of chemiluminescent signal intensity between CNN1 and loading control ACTB are shown.

Fig 3: A: Mitochondrial localization of COQ8B by PK assay. Immunoblots of COQ8B-HA compared with MOM marker TOM20 and mitochondrial matrix marker GPR75 from enriched mitochondrial fractions of HEK293 cells. B: Carbonate extraction experiments to determinate COQ8B association to membranes. Immunoblots of COQ8B were compared with SCO2 and porin (integral membranes proteins), SDHA (a peripheral MIM protein) and cytochrome c (a soluble protein). P, pellets; S, soluble fraction. C: Human COQ8B cannot complement ?COQ8 yeast strains. D: Structure of the different COQ8B constructs used in this work. COQ8B amino acids 36–54 were not conserved during evolution. E: Both hybrid constructions yCOQ8-COQ8B and yCOQ3-COQ8B can complement ?COQ8 yeast even when expressed from a low-copy centromeric vector. F: Coenzyme Q6 levels and (G) complex II + III activity normalized to citrate synthase (CS) in complemented yeast strain. ND, not detectable. H: Anti-V5 immunoblot of ?COQ8 yeast expressing COQ8B, yCOQ3-COQ8B or the empty vector. For the last three experiments, yeast strains were grown in YPGly medium supplemented with 0.1% glucose. Porin was used as loading control

Fig 4: Oxidative stress and mitochondrial damage in the proximal aorta in TAA(A) Semiquantitative scoring of the intensity levels of intracellular anti-nitrotyrosine staining in the aortic medial layer between TAA and controls. Groups were compared using Cochran-Armitage test.(B) Example of heavy anti-nitrotyrosine staining intensity in the ascending aortic medial layer in TAA compared with control aorta. Blue staining labels nuclei and brown staining labels nitrotyrosine. Black arrow indicates a cell with heavy staining.(C) Scoring of mitochondrial cristae defects between TAA (N = 7 individuals) and control (N = 3 individuals) aortic tissues imaged by transmission electron microscopy. Each point represents the mean score per individual. A median 79 (interquartile range [IQR], 55 to 117) mitochondria were analyzed per individual.(D) Examples of normal mitochondria in controls (green arrows) and enlarged mitochondria with abnormal cristae in TAA (red arrows). Yellow bar indicates 250 nm.(E) Levels of H2O2 in cultured aortic SMCs from TAA (N = 2 individuals) and control aorta (N = 2 individuals) measured by Amplex Red fluorescence intensity indexed for cell confluency. Each point is the result of an independent well, and bars indicate mean for each individual.(F) RNA levels of COQ8B between TAA (N = 7 cases) and controls (N = 4 individuals) in cultured proximal aortic SMCs. COQ8B RNA levels were adjusted for ACTB and are displayed relative to mean of control SMCs. Each point corresponds to the mean of three independent wells of SMCs. Replicate values are shown in Figure S4. TAA and control groups were compared using a Student’s t test.*p < 0.05.

Fig 5: Mutated COQ8B residues modeled into the three-dimensional structure of COQ8A (PDB code 4PED)