(A) Relative quantification of the endogenous COL6A6 mRNA in two UCMD and four BM patients. The COL6A6 gene is up-regulated in all patients (fold change ranging from 3.297 up to 171.254). The COL6A6 mRNA level in control myoblasts (CTRL) is set to 1. The values reported in the graph are the average between two independent experiments. (B) Immunofluorescence analysis of the α1α2α3 heterotrimer in control, four BM and two UCMD-confluent muscular fibroblasts after 4 days of l-ascorbic acid treatment. The α1α2α3 chains appeared normally assembled and secreted in BM-2 and BM-6 cultures, while they were reduced in BM-1 and in all UCMD patient cultures. A particularly strong intracellular retention was detected in the UCMD-2 patient (inset). Nuclear staining, DAPI. Scale bar: 20 μm. (C) Western blot analysis of cell layer from muscle cell cultures derived from two unaffected donors (CTRL), and BM-1, BM-2, BM-6, UCMD-2, UCMD-6 and BM-8 patients. Cells were treated for 4 days with l-ascorbic acid post-confluence, scraped and samples were separated on a 4–20% polyacrylamide gel under reducing conditions. The α1α2α3 heterotrimer and the α6 chain were evaluated by specific antibodies. The loading control was performed using a GAPDH antibody. Lower numbers indicate α6 chain/GAPDH ratio by densitometry. (D) Western blot analysis of cell layers from two unaffected donors (CTRL), and BM-1, BM-2, BM-6, UCMD-2, UCMD-6 and BM-8 patients treated for 4 days with l-ascorbic acid and 10 ng/ml TGF-β1. Cells were scraped and samples were separated on a 4–20% polyacrylamide gel under reducing conditions. The α1α2α3 heterotrimer and the α6 chain were evaluated by specific antibodies. GAPDH was used as a loading marker. Lower numbers indicate the α6 chain/GAPDH ratio by densitometry. (E) Immunofluorescence analysis of the α6 chain in control (CTRL), BM-1, BM-2, BM-6, UCMD-2, UCMD-6 and BM-8 patient muscle cell cultures treated for 4 days with TGF-β1 and l-ascorbic acid. All patients’ cells displayed a strongly reduced amount of the α6 chain, with a significant intracellular retention in BM-1 and BM-6 muscle cells. Nuclear staining, DAPI. Scale bar, 10 μm.