Fig. 2

mtDNA variants affect mitochondrial ultrastructure and OXPHOS complexes activity and abundance. A Representative western blot analysis and relative protein expression of mitochondrial complexes subunits (UQCRC2, SDHB, COXII, NDUFB8) in PDOVCAs. GADPH was used as loading control and to perform densitometric normalization. B Representative images and relative quantification of MT-CO1 protein expression were obtained by IHC in PDX cells. The percentage of 3 + 2 + positive cells was measured through ImageJ. Scale bar 100 µM. C Activity of mitochondrial respiratory complexes determined by spectrophotometric kinetic measurements and normalized to protein concentration in homoplasmic (VAF = 100%, PDOVCA 5, 62, 126) and heteroplasmic (VAF < 100%, PDOVCA 69 and 49) PDOVCAs. Data (n ≥ 3) are mean ± SD and expressed as percentage relative to controls (wild type and mutated PDOVCA with heteroplasmy < 25%). *p < 0.05, **p < 0.01, ***p < 0.001, according to ordinary one-way ANOVA. D Representative TEM images of mutated (MUT: PDOVCA 5, 62) and wild-type (WT: PDOVCA 15, 17) PDOVCA-derived cells. Cells were collected, fixed, embedded, sectioned and viewed using TEM. Mutated mitochondria were visualized and appeared morphologically altered respect to wild-type ones. Scale bar 2 µM. E Mitochondrial number and area were calculated from TEM images (n = 50) in MUT and WT PDOVCAs. Both parameters were obtained normalizing the number of mitochondria and the occupied area on cytosol area in each image. F Mitochondrial ROS in MUT and WT PDOVCAs. Data (n ≥ 3) are mean ± SD (n = 3 biological replicates for each PDOVCA). **p < 0.01, ***p < 0.001, according to unpaired Student’s t test (two-tailed)