Fig. 2: Muscle histology and biochemical studies in myoblasts of the COX18-mutated patient.

A Modified Gomori Trichrome B Severe reduction of muscle fibers staining positive for cytochrome c oxidase (COX) is seen in the COX18 subject, whereas normal COX staining is shown in a healthy control (C) for comparison (Scale bar 25 µm). D–F Representative images of the alterations observed in mitochondria. Inner cristae were arranged to form concentric layers. (Asterisk in F indicates an osmiophilic inclusion. Scale bars D = 926 nm, E–F = 463 nm). G Spectrophotometric analysis of respiratory chain complexes activities normalized to citrate synthase levels in patient’s and control myoblasts. H Immunoblot analysis of myoblasts protein with antibodies directed against respiratory chain subunits. The mitochondrial porin (VDAC) and the cytosolic Actin proteins were used as a loading control. Densitometry (right) shows the reduction of steady state levels of COX subunits COX-II, COX-III and COX-IV in patient’s compared to controls’ myoblasts. I Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis of myoblasts protein shows absence of fully assembled COX in the COX18 subject. Each of the five OXPHOS complexes (I–V) was visualised with a subunit-specific antibody that recognizes the native complex as follows: Complex I (NDUFA9), Complex II (SDHA), Complex III (UQCRC1), Complex IV (COX4), Complex V (ATP5A1). Densitometry after SDHA normalization (right) shows the prevalent reduction of fully assembled Complex IV. J In-gel activity assay showing the severe reduction of Complex IV activity in patient’s cells compared to controls.