Impaired autophagy, increased polyubiquitination and dysfunctional mitochondria in human MPS VI fibroblasts. (A) Protein extracts from fibroblasts of three controls (NR fibroblasts) and of seven MPS VI patients (MPS VI fibroblasts) were assessed for glycosaminoglycans (GAGs) accumulation by the quantitative dimethyl-methylene blue method (GAG levels are expressed as μg per mg of proteins: μg GAG/mg prot). (B and C) Representative western blot analysis of total lysates from NR and MPS VI fibroblasts blotted with anti-BCN1, -LC3, -p62, -COX IV (B) and -ubiquitin antibodies (C). Normalization of protein loading was performed using anti-actin antibodies (B) and is the same for both panels B and C. (D) Quantification of western blot analyses from three independent experiments (mean ± standard error). Values are expressed as fold of increase compared with NR fibroblasts (NR = 1). (E) Confocal images of NR and MPS VI fibroblasts stained with anti-LC3 (green) and anti-LAMP2 (red) antibodies. Inserts illustrate the colocalization of the two proteins (merge panel). Magnification: 63×. (F) Clearance of loaded Alexa Fluor 488-labeled EGF at various time points (T0, T30, T60, T120 and T180 = 0 min, 30 min, 1 h, 2 h, and 3 h after loading, respectively). Magnification: 100×. (G) Human NR and MPS VI fibroblasts were labeled with anti-ubiquitin, -p62 and -COX IV antibodies. Magnification: 63×. (H) Measurement of mitochondria membrane potential by flow cytometry analysis of NR and MPS VI fibroblasts after loading with DiOC6 and propidium iodide (PI). All experiments were performed in triplicate. *P ≤ 0.05, **P ≤ 0.02, ***P ≤ 0.01.