Samples
In all experiments we used immortalized myoblasts from a dysferlinopathy patient and from a healthy control. The patient, number 16 in our previous study, had homozygous missense mutations (c.4882G>A/p.G1628R) in exon 44 of DYSF and sarcoplasm showed residual dysferlin of about 1-2% compared to WT myotubes [24]. These cells were immortalized and kindly provided by Dr Mouly [25]. Myoblasts were expanded using skeletal muscle medium (SMM; Promocell, Heidelberg, Germany) until confluence was achieved. The media was then changed to differentiation media (75% Dulbecco’s Modified Eagle’s Medium and 25% M199, supplemented with 2% FBS (Lonza, Basel, Switzerland), 10 μg/ml insulin (Sigma-Aldrich, St Louis, MO, USA), 2 mmol/l glutamine (Lonza) and penicillin-streptomycin (Lonza)) for 7 to 9 days. Differentiated myotubes were treated with vitamin D3 or EB1089 (Sigma Aldrich) at 100 nM, ixazomib (Selleckchem, Munich, Germany) (25nM, 50nM and 100nM) and oprozomib (ONX 0912)(Selleckchem) (10nM, 50nM and 100nM). Ixazomib and oprozomib were combined with EB1089 at 8h and 24h, respectively.
Western Blot
Cell pellets corresponding to each condition were lysed in RIPA buffer (Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Lysates were centrifuged at 4 °C at 13000 x g for 20 min and supernatants were stored at −80°C. Protein concentrations were determined using Pierce™ BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA). Thirty µg of protein were resolved in a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to nitrocellulose membranes. Non-specific binding sites were blocked by incubation for one hour in casein diluted 1:1 in tris-buffered saline (TBS). Blots were incubated overnight with the primary mouse monoclonal antibodies anti-dysferlin (NCL-Hamlet, Novocastra, Newcastle, UK) and anti-myogenin (5FD clone) (Santa Cruz Biotechnology, Dallas, TX). To normalize the results, mouse anti-desmin (Novocastra) was added simultaneously with the primary antibody. The secondary antibody for anti-dysferlin was a goat anti-mouse labeled with IR-Dye 800 (Li-Cor, Lincoln, Nebraska, USA). When the anti-myogenin antibody was used as a primary antibody, biotin-labelled horse anti-mouse (Jackson ImmunoResearch, Ely, UK) was used as a secondary antibody. Secondary antibodies were incubated for one hour at room temperature. Membranes incubated with biotinylated secondary antibody were washed and incubated for one hour with IRDye-680-labelled streptavidin (Li-Cor). After extensive washing, the immunoreactive bands were visualized using the Odyssey Infrared ImagingSystem (Li-Cor). The amount of protein was quantified using Image Studio Lite software (Li-Cor). Desmin expression was used as a loading control. As a reference value we used dysferlin or myogenin expression in the healthy control. Protein expression in the remaining conditions was quantified over the expression in WT myotubes and expressed as fold-change.
Assessment of proteasome-like activity
Cells were seeded in a 96-well black plate (Sarstedt, Nümbrecht, Germany) at 5000 cells/well in three replicates until confluence. The media was then changed to differentiation medium. Cells were treated with the corresponding drug for the indicated time. CT-L, C-T and T-L activity was assayed by chemiluminiscence using the Proteasome-Glo™ 3-Substrate System cell based assay (Promega, Madison, WI, USA ) and the plate was read using Victor 3v Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA).
Thrombospondin-1 Enzyme-Linked Immunosorbent Assay (ELISA)
Immortalized myoblasts from the dysferlinopathy patient were seeded at 5000 cells/cm2 and expanded until confluence. The media was then changed to differentiation medium to form myotubes. After treatment with proteasome inhibitors together with or without EB1089, the media was removed and added 1 ml of basal DMEM (Lonza) was added to the culture for 24 hours. Cell culture supernatants were concentrated using Amicon Ultra Centrifugal Filters 100 kb (Merck Millipore, Darmstadt, Germany). TSP-1 present in the culture media was detected using the human TSP-1 Immunoassay (Quanti-Kine ELISA, R&D Systems, Minneapolis, MN), following the manufacturer’s instructions. The detection limit of the assay was 0.355 ng/mL.
Membrane Repair
Assay
Immortalized myoblasts from the dysferlinopathy patient and the healthy control were grown until confluence in chambered wells and then differentiated to myotubes. The assay was performed as recently described in human muscle primary cultures [26]. SDS is an anionic surfactant that causes the leakage of intracellular components due to its ability to affect membranes [27]. Membrane injury was induced by detergent treatment as follows: after the cell cultures were washed with Hank’s Balanced Salt Solution (HBSS) (Lonza), the injury solution (HBSS with 0.12mM or 0.25mM SDS (Sigma-Aldrich)) was applied for two minutes. Following exposure to injury solution, cells were washed in HBSS and then incubated in recovery solution consisting of proliferation media for 90 seconds and 10 minutes. The injury and recovery steps were performed at 37ºC. Cells with injured permeable membranes were identified by exposure to propidium iodide (PI) (Sigma-Aldrich) (20 µg/mL in HBSS). If cell membrane is not repaired, PI enters the cell and penetrates the nucleus wherein it intercalates with DNA and emits fluorescence. After two minutes of exposure to the dye, cells were washed with HBSS and fixed with 4% paraformaldehyde in PBS. Nuclei were stained with Hoescht 33342 (Invitrogen, Thermo-Fisher Scientific). In each experimental condition, we counted the total number of nuclei and the PI positive nuclei using Fiji software [28].
Fusion index
Following the membrane repair assay, myotubes were permeabilized with ethanol for five minutes at room temperature. Samples were then blocked with the UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Dallas, TX) for one hour at RT. Myotubes were stained using a mouse anti-myosin heavy chain (MyHC) antibody (MF-20)(Bio-Rad, Hercules, CA, USA) for one hour at RT. The goat anti-mouse secondary antibody Alexa Fluor 594 (ThermoFisher, Wal- tham, MA) was then used as a secondary antibody for one hour at RT. Following incubation with the secondary antibody, nuclei were stained with Hoescht 3342 (Invitrogen, Thermo-Fisher Scientific). Images were acquired and analysed using Fiji software.
The fusion index was determined as the percentage of nuclei included in MF-20-expressing myotubes (containing at least three nuclei) divided by the total number of nuclei.
Statistical Analysis
Multiple comparisons were analyzed using a one-way ANOVA test followed by Tukey post hoc test. When statistical comparisons were performed between two groups, the nonparametric Mann-Whitney test was used. GraphPad Prism 5.0 software was used (LaJolla, CA, USA) for graphic representation.