*4.2. Generation of DG-KO C2C12 Cell Lines by CRISPR-Cas*

Two different single guide RNAs were designed to target the first coding exon of *Dag1* gene, using the crispr.mit.edu online tool: gRNA1 (5' CCGACAACAGCCGTACCGTC 3') and gRNA2 (5' CCAGACGGTACGGCTGTTGT 3') gRNAs were cloned into pSpCas9(BB)-red fluorescent protein (RFP) plasmid (modified from pSpCas9(BB)-2A-GFP, a gift from Feng Zhan–Addgene plasmid # 48138; http://n2t.net/addgene:48138;RRID:Addgene\_48138), Addgene (Watertown, MA, USA). C2C12 cells were transfected with gRNA1- or gRNA2-Cas9-RFP plasmids using lipofectamine (LTX) with plus reagent (Thermo Fisher Scientific, Waltham, MA, USA). Forty-eight hours post transfection, RFP positive cells were sorted using FACSAria (BD Biosciences, Woburn, MA, USA). After expansion, cells were collected with enzyme-free cell dissociation buffer (Gibco–Thermo Fisher Scientific, Waltham, MA, USA) and incubated with anti-CD16/CD32 antibody (Mouse BD Fc Block, 2.4G2; BD Biosciences, Woburn, MA, USA) on ice for 5 minutes, and subsequently with anti-α-DG antibody (IIH6C4) on ice for 30 minutes. Following PBS washes, cells were incubated with goat anti-mouse IgG-Alexa Fluor 488 secondary antibody on ice for 20 minutes. Cells were then washed with PBS and resuspended in FACS buffer (10% FBS in PBS). Cells negative for α-DG staining were sorted by FACS and expanded. WT cells incubated with or without IIH6C4 were used to set the gates for positive or negative α-DG staining, respectively. Upon expansion, sorted cells had a second round of sorting for α-DG negative staining and single cells were collected in a 96-well plate for clonal expansion. DG-KO clones were screened for β-DG by Western blotting using anti-β-DG antibodies (MANDAG2). Two clones, DG-KO1 and DG-KO2, were expanded and characterized by sequencing the DNA region targeted by the gRNAs, to confirm *Dag1* gene disruption. Cell cultures between passage six and twelve were used for all analyses.

#### *4.3. Antibodies*

The following primary antibodies were used. Mouse monoclonal antibodies againstα-dystrobrevin (α-DB; BD Transduction Laboratories, Becton Dickinson, Franklin Lakes, NJ, USA), α-DG (IIH6C4 (IIH6, 05-593; Millipore, Sigma-Aldrich, St. Louis, MO, USA), β-DG (MANDAG2 [57]), α-tubulin (sc-32293; Santa Cruz Biotechnology, CA, USA), p53 (#2524; Cell Signaling Technology, MA, USA), p21 (#2946; Cell Signaling Thecnology, MA, USA), and GAPDH (sc-32233; Santa Cruz Biotechnology, CA, USA). Rabbit polyclonal antibodies against B23 (sc-6013-R; Santa Cruz Biotechnology, CA, USA), dystrophin Dp71 (+78Dp71; Genemed Synthesis Inc., San Francisco, CA, USA), lamin B1 (Ab16048; Abcam, Cambridge, UK), γ-tubulin (sc10732; Santa Cruz Biotechnology, CA, USA), H3K9me3 (ab8898; Abcam, Cambridge, UK) and γ-H2AX (#07-164; Millipore, Sigma-Aldrich, St. Louis, MO, USA). Goat polyclonal antibody against β2-syntrophin (SC-13766; Santa Cruz Biotechnology, CA, USA) was also used.

#### *4.4. Western Blotting*

C2C12 cell culture lysates were electrophoresed on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories Inc., Berkeley, CA, USA). Membranes were blocked in TBST (100 mM Tris-HCL pH 8.0, 150 mM NaCL, 0.5% (*v*/*v*) Tween-20) with low fat-dried milk and then incubated overnight at 4 ◦C with the appropriate primary antibodies. The specific protein signal was developed using the corresponding secondary antibodies and enhanced chemiluminescence western blotting detection system (ECL TM; Amersham Pharmacia, GE Healthcare), according to the manufacturer´s instructions. Images were acquired for densitometric analysis with a Gel Doc EZ System (Bio-Rad Laboratories Inc., Berkeley, CA, USA), using Image Lab 6.0.1 software (Bio-Rad Laboratories Inc., Berkeley, CA, USA). To normalize protein expression from the same sample and on the same blot, the band intensity of the target protein was divided by the band intensity of the loading protein.
