Utrophin

Each experiment was performed by several operators and different operators were arbitrarily assigned to each of the subsequent processing steps (sectioning of muscle biopsies, staining of sections for dystrophin and spectrin, image acquisition by confocal microscopy, and software-assisted image analysis). Biopsy quality was assessed by hematoxylin and eosin staining and was routinely performed to evaluate freezing artefacts (biopsy handling, transport and storage) and relative amount of fibrotic and adipose tissue, and to ensure sufficient fibers for reliable IFA for dystrophin.
Using a cryotome (Thermo Fisher Scientific, Waltham, MA, USA), 8 µm muscle cross-sections were cut and placed on Superfrost Ultra Plus Microscope Slides (Thermo Fisher Scientific, VWR 631–0099). After drying at room temperature for 1 hour, the slides were placed in a −80°C freezer and used within 2 months. The staining procedure was performed at room temperature. Slides were removed from the freezer, air dried for 20 minutes, fixed with acetone for 1 minute, washed with PBS, blocked with PBS containing 0.05% Tween-20 and 5% horse serum for 1 hour, and subsequently washed with PBS. For each biopsy, two or four sections were stained. Double-staining for dystrophin and spectrin was performed with the following combination of antibodies: mouse monoclonal MANDYS106 rod-domain anti-dystrophin antibody [9] (link), [10] (link) combined with a rabbit anti-spectrin antibody, or rabbit polyclonal ab15277 C-terminal anti-dystrophin antibody combined with a mouse anti-spectrin antibody (Table 2). Cross-reactivity to utrophin has been a concern for C-terminal anti-dystrophin antibodies, but is highly unlikely for ab15277 as it is raised to a peptide epitope unique to dystrophin and is affinity purified. Sections were incubated for 2 hours with anti-dystrophin or isotype antibody alone followed by a 1-hour incubation with anti-dystrophin and anti-spectrin antibodies combined, or isotype and anti-spectrin antibody combined. After the incubation with the primary antibodies, the sections were washed with PBS and then incubated with the appropriate secondary antibodies combined for 1 hour (Table 2). After washing again with PBS, the slides were mounted with Vectashield Mounting medium (Vector Laboratories, Burlingame, CA, USA; Brunschwig 6-H-1400) and imaged on the same day (Table 2).

Histopathological and immunofluorescent characterizations of muscle biopsies from both Brittany spaniels were previously described [30 (link)]. Unfixed, chilled and formalin-fixed diagnostic muscle biopsy specimens were collected post-mortem from the biceps femoris, diaphragm and tongue muscles of the French bulldog and shipped by an overnight express service under refrigeration to the Comparative Neuromuscular Laboratory, University of California San Diego. Upon receipt, the unfixed muscle specimens were snap frozen in isopentane, pre-cooled in liquid nitrogen, and stored at −80 °C until further processed. Cryosections were evaluated using a standard panel of histochemical stains and reactions [31 ]. Additional cryosections were cut and stained for indirect immunofluorescence as previously described [32 (link)], using monoclonal antibodies against the rod (1:100, NCL-DYS1) and carboxy-terminus (1:100, NCL-DYS2) of dystrophin, utrophin (1:20, NCL-DRP2), and developmental myosin heavy chain for regenerating fibers (1:20, NCL-dMHC), all from Novocastra Laboratories, Newcastle, UK; a monoclonal antibody against caveolin 3 (1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and polyclonal antibodies against laminin α2 (1:200), α-sarcoglycan (1:200), and collagen VI (direct apply, monoclonal antibody 3G7), all gifts of Professor Eva Engvall [33 (link),34 (link)].

RNA was isolated from muscle tissues or cultured cells with TriPure reagent (Sigma-Aldrich). RT-qPCR primers for mouse cyclophilin, Collagen Type I Alpha 1 Chain (COL1A1), Collagen Type III Alpha 1 Chain (COL3A1), oestrogen receptor-related alpha (ERRα), Myogenic regulatory factors 4 (Mrf4), Myh1 and Myh7 are provided in Table S2. RT-qPCR primers for human TATA box-binding protein (TBP), AdipoR1, IL-1β, TNFα and Utrophin (UTRN) are also indicated in Table S2. Threshold cycles (Ct) were always measured in duplicate.