Fig 2: Corrector C17 induced a time dependent increase of mutated a-SG, without toxicity, in LGMD2D myotubes. (A) myogenic cells from a patient carrying the L31P/V247M a-SG mutations were grown and differentiated for 7 days and treated with 1‰ DMSO (vehicle) or 15 µM C17 for the indicated time intervals. a-SG protein content was evaluated by western blot of total myotube lysates. The Ponceau red staining (PR) is reported to normalize the total amount of proteins loaded in each lane. (B) quantification by densitometric analysis of a-SG protein bands of three independent western blot experiments, as described in (A). The average amount of a-SG (± SEM) is expressed as fold increase of the protein content present in myotubes treated with vehicle for the same incubation interval. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Bonferroni test; *P = 0.05; ***P = 0.001. (C) phase contrast images of myotubes treated with either 1‰ DMSO (vehicle) or C17 at the concentration and time intervals indicated to evaluate possible toxic effects. All images were recorded at the same magnification.

Fig 3: Additive effect of C13 in combination with C17. Myogenic cells from a patient carrying the L31P/V247M a-SG mutations were differentiated for 7 days and treated for the last 72 h with either 1‰ DMSO (vehicle), C13, C17 or C13 + C17 at the indicated concentrations. At the end of incubation, surface proteins were biotinylated. Then, myotubes were lysed and, after quantification, 50 µg of proteins were subjected to pull down assay by streptavidin-conjugated agarose beads. Recovered surface proteins and 1/10 of the starting myotubes lysates (input) were analyzed by SDS-PAGE and western blot with antibodies against a-SG and the cytosolic protein ß-actin used as loading control (western blot of input) and to check the absence of biotin internalization (western blot of biotinylated proteins). (A) Phase contrast image showing the LGMD2D myotubes at the end of the combined treatment. Scale bar corresponds to 100 µm. (B) Representative western blot in which the left part shows the immunodetection of a-SG and ß-actin present in the input lysates and the right part the fraction present at the sarcolemma; under the blot, the Ponceau Red staining of the membrane is reported as control of protein loading. Representative western blots of myotubes treated with vehicle, C17 10 µM or 15 µM is reported in Figure S2. (C) Quantification of a-SG content by densitometric analysis of the input and (D) of the biotinylated fraction of proteins. Values are the mean (+/- SD) of 3–4 independent experiments (each performed in duplicate) and are reported as fold increase of the amount present in the control sample. Statistical analysis was performed by One-way ANOVA test followed by multiple comparisons Tukey’s test; n.s., p > 0.05; *, p = 0.05; **, p = 0.01; ***, p = 0.001, ****, p = 0.0001.

Fig 4: Rescue of a-SG by cystic fibrosis transmembrane regulator (CFTR) correctors in LGMD2D myotubes. Myogenic cells from a patient carrying the L31P/V247M a-SG mutations were differentiated for 7 days and treated for the last 72 h with either 1‰ DMSO (vehicle), C13, C9, C6 or C17 at the indicated concentrations. (A) phase contrast images of myotubes at the end of corrector incubation are indistinguishable from the control ones. Scale bar corresponds to 100 µm. (B) Densitometric analysis of western blot experiments performed with total protein lysates from LGMD2D myotubes treated as indicated. The a-SG protein was revealed by using specific primary antibody and normalized by the content of ß-actin. a-SG content in different samples was expressed as the fold increase (+/- SD) of the amount present in the vehicle treated myotubes, and is the mean value of 3-4 independent experiments (each performed in duplicate). Statistical analysis was performed by One-way ANOVA test followed by multiple comparisons Dunnett’s test; n.s., p > 0.05; ***, p = 0.001; ****, p = 0.0001.

Fig 5: The LGMDR3 mouse model with humanized hind-limbs. (A) Scheme of the experimental work flow (created with BioRender.com). (B) Histological (H&E) and IF analyses of TA muscles cryo-sections from C57BL/6 (positive control), sgca-null (negative control) and sgca-null transduced with either the human WT a-SG sequence (sgca-null + h-a-SG-WT, positive control of transduction), the human V247M-a-SG sequence (sgca-null + h-a-SG-V247M) or the human R98H-a-SG sequence (sgca-null + h-a-SG-R98H). Primary antibodies specific for a-, d- and ß-SG, as indicated, were revealed by Alexa-Fluor488-conjugated secondary antibodies. IF images were captured at the same setting conditions. Bars correspond to 100 and 50 µm in H&E and IF images, respectively. (C) WB analysis of total protein lysates of representative TA muscle samples from negative and positive control (sgca-null and C57BL/6, respectively) and mice humanized with the different a-SG sequences, as indicated. The membrane was probed with primary antibodies to a-SG and GAPDH, used as loading control together with Ponceau Red protein staining. Two exposition-times of the a-SG-probed membrane are reported to appreciate the expression of the R98H mutant.