293 T cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, D6546), supplemented with 10% fetal bovine serum (Gibco, 10270), 2 mM l-glutamine, and 1% penicillin/streptomycin solution (Sigma-Aldrich, G7513; P0781) at 37°C under 5% CO2. Murine myoblast C2.7 cells (61), rat myoblast L6E9 cells (62), and human fibroblast–derived myoblast cells from an HD and a patient with LGMD2H were cultured in DMEM, supplemented with 20% fetal bovine serum, 2 mM l-glutamine, and 1% penicillin/streptomycin solution at 37°C under 5% CO2. Myoblast differentiation was induced by culturing cells in DMEM supplemented with 2% horse serum (Life Technologies, 16050122), 2 mM l-glutamine, and 1% penicillin/streptomycin for an indicated amount of time. Fibroblasts from an HD and a patient with LGMD2H were provided by the Telethon Network of Genetic Biobanks project no. GTB12001. To prepare fibroblasts, a patient’s skin biopsy was obtained after informed consent and approval of the Ethics Committee of the University of Ferrara. No cell lines used in this study were found in the database of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee and National Center for Biotechnology Information biosample. Cells were screened for mycoplasma contamination by PCR (ABMgood, G238).

To evaluate autophagy, C2.7 cells and HD/LGMD2H-differentiated myoblasts were treated with 400 μM dexamethasone (Calbiochem, 265005) in the presence or absence of E64d/pepstatin A (5 μg/ml) (Santa Cruz Biotechnology, sc-201280A and sc-45036) or 5 nM Bafilomycin A1 (Sigma-Aldrich, B1793) or incubated with nutrient-deprived medium Earle's balanced salt solution (Sigma-Aldrich, E2888) for the indicated time, according to guidelines. The autophagy inhibitor 3MA (Sigma-Aldrich, M9281) was used at 5 mM.

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