Fig 2: Dystrophin localizes to the centrosome.(A) Double immunocytochemistry (ICC) of C2C12 myoblasts using antibodies specific for dystrophin (red) and γ-tubulin (green) and visualization of nuclei (DAPI, blue). Insets are magnifications of the centrosomes (as indicated by the boxes I and II). Note the co-localization of dystrophin with γ-tubulin. (B) Fluorescence intensity plots illustrating the respective distribution of dystrophin (red) and γ-tubulin (green). The white dashed line denotes the direction of the profiling through the centrosome (shown in inset I in A). Note that dystrophin overlaps the γ-tubulin-positive area. a.u., arbitrary units. (A, C) ICC of proliferating C2C12 myoblasts as in (A) at different phases of the cell cycle. (A, D) ICC of primary murine and human myoblasts as described in (A). (E) Co-purification of dystrophin with γ-tubulin in centrosome-enriched subcellular fractions. Centrosomes were purified from C2C12 myoblasts by sucrose density-gradient centrifugation. Fractions were analyzed by immunoblotting. Note that in centrosome-enriched fractions 4, 5, and 6, as evaluated by increased γ-tubulin protein levels, also full-length dystrophin (arrowheads) was detected. (F) ICC of murine (p53−/−) and human (WI-38) fibroblasts using antibodies specific for dystrophin (red) and γ-tubulin (green) and visualization of nuclei (DAPI, blue). Insets are magnifications of the centrosomes (as indicated by the boxes I, II, and III). Scale bars (A, C, D, F), 5 μm. (G) Expression of dystrophin in murine skeletal-muscle lysates and cell lysates prepared from mouse myoblasts (C2C12) and fibroblasts (p53−/−). (H) Expression of dystrophin in human skeletal-muscle lysate and cell lysate prepared from human fibroblasts (WI-38). GAPDH (G, H), loading control. (I) Scheme illustrating the nested RT–PCR strategy used to detect dystrophin transcripts starting from the dystrophin muscle promoter (muscle-specific exon 1) extending into exon 11. As full-length dystrophin (Dp427) isoforms start from individual promoters (cortical/brain, muscle, or Purkinje cell promoter) into unique first exons followed by a common exon 2 (Muntoni et al, 2003; Doorenweerd et al, 2017); this strategy enabled the specific detection of dystrophin transcripts expressed from the muscle-specific promoter only. Moreover, as shorter dystrophin isoforms usually lack the amino-terminal dystrophin amino acids, RT–PCR products obtained by this strategy are likely to correspond to full-length Dp427m. (J) Representative results of nested RT–PCR for mouse dystrophin. Note that for mouse fibroblasts, in addition to the expected 1,113-bp PCR product (arrowhead), a second band of 981 bp was observed (arrow), corresponding to a transcript lacking exon 9 (Reiss & Rininsland, 1994). (K) Representative results of nested RT–PCR for human dystrophin. Note that for HeLa cells, in addition to the expected 1,024-bp PCR product (arrowhead), a second band of 895 bp was observed (arrow), corresponding to skipping of exon 9 (Reiss & Rininsland, 1994) in this cell line as well. NTC (J, K), no template control. The specificity of all PCR products in J and K was verified by DNA sequencing.Source data are available for this figure.

Fig 3: Thbs4 is induced in dystrophic skeletal muscle and its overexpression augments ER activity and vesicle content.(A) Thbs4 mRNA levels in human skeletal muscle biopsies from normal or patients with Becker MD (BMD; n = 5), Duchenne MD (DMD; n = 10) or 2 different types of limb-girdle MD (LGMD; n = 10 for both). *p<0.05 vs. normal (n = 18) by Student’s t test. Data are presented as mean ± SEM. Full analysis including all 11 human muscle diseases is shown in Figure 1—figure supplement 1A. (B,C) Western blot for the expression of Thbs4, ATF6a-N (50 kDa, nuclear) and BiP in the quadriceps of WT, Sgcd-/- and mdx mice at six weeks (w) and three months (mo) of age (n = 4 biological replicates). (D) Schematic diagram showing the skeletal muscle-specific transgene to overexpress Thbs4 and (lower) Western blots for Thbs4 or gapdh control from WT and Tg mice at 6 w of age from Quad, quadriceps; Gas, gastrocnemius; Sol, soleus; Diaph, diaphragm; and heart (n = 2 biological replicates). (E) Upper micrographs represent co-immunofluorescent labeling of intracellular Thbs4 (green) with calreticulin (red) on paraffin embedded quadriceps (Quad.) of WT, Thbs4-Tg and Sgcd-/- mice at 3 mo of age (scale bar = 20 µm). Arrowheads indicate co-localization of Thbs4 with calreticulin in intracellular vesicles in the myofibers. Lower micrographs represent co- immunofluorescent labeling of Thbs4 (green) with collagen I (red) on cryo-embedded Quad of WT, Thbs4-Tg and Sgcd-/- mice at 3 mo of age (scale bar = 50 µm). Arrowheads indicate co-localization of Thbs4 with collagen I in the extracellular milieu; the star marks a myofiber with both intra- and extracellular Thbs4 labeling from a diseased muscle. Nuclei are visualized in blue. Representative images of 4 mice per genotype are shown. (F) Western blot analysis of Thbs4 and the ER-stress proteins ATF6a-N (50 kDa, nuclear), BiP, PDI, calreticulin (Calret.), and Armet in 6w old WT, Thbs4-Tg and Sgcd-/- quadriceps (n = 4 biological replicates). (G) Transmission electron microscopy in WT versus Thbs4 Tg quadriceps at 3 mo of age showing a massive expansion of intramyofibrillar and subsarcolemmal ER and associated vesicles with Thbs4 overexpression (arrowheads, scale bar = 2 µm). Representative images of 2 mice per genotype are shown. (H) Immunogold electron microscopy shows that Thbs4 (6 nm gold particles; yellow arrows) robustly localizes to the expanded sub-sarcolemmal vesicular compartment in Thbs4-Tg quadriceps, compared to endogenously expressed Thbs4 in WT quadriceps. Representative images of 2 mice are shown. Scale bar = 50 nmDOI: http://dx.doi.org/10.7554/eLife.17589.003

Fig 4: NFA and LPA treatments enhance the engraftment efficiency of mouse satellite cells(A) Experimental design.(B and C) Engraftment efficiency of 4-h-treated mouse satellite cells with vehicle, LPA (5 μM), or NFA (10 μM). Five thousand treated cells from FVB-Tg(CAG-eGFP) mice were injected into pre-injured TA muscles of non-transgenic FVB/NJ recipient, and engraftment was measured by counting the number of GFP+ myofibers. Data are presented as mean ± SEM. Statistical analysis by unpaired t test; both treatment groups were significantly different from the control group (n = 10).(D) Transverse frozen section of TA muscles injected with 5,000 treated satellite cells. Cell membrane (WGA, red), nuclei (DAPI, blue), and engrafted cells (GFP, green). These studies used to 16-week-old male mice as recipients. DMSO concentration for all groups including experimental control and as vehicle for NFA and LPA was 0.1%. Scale bar, 200 μm.(E) Engraftment efficiency of 4-h-treated mouse satellite cells with vehicle, LPA (5 μM), or NFA (10 μM). Two thousand treated cells from C57BL/CAG-EGFP mice were injected into pre-injured TA muscles of PrkdcscidDmdmdx recipients, and engraftment was measured by counting the number of GFP+ myofibers. Data are presented as mean ± SEM. Statistical analysis by one-way ANOVA; both treatment groups were significantly different from the control group (n = 9 for vehicle and n = 7 for LPA and NFA treatment groups).(F) Restoration of dystrophin in engrafted PrkdcscidDmdmdx recipients using a mouse-specific dystrophin antibody (Sigma, D8043). Nuclei (DAPI, blue), engrafted cells (GFP, green), and dystrophin (Alexa Fluor 594, red). Scale bar, 50 μm.(G) NFA and LPA treatments do not change the rate of incorporation of transplanted satellite cells into the in vivo pool of muscle precursors. Donor mouse satellite cells were isolated from Pax7:ZsGreen mice and treated for 4 h with vehicle (0.1% DMSO), LPA (5 μM), or NFA (10 μM). Five thousand treated cells were transplanted into pre-injured TA muscles of WT (C57BL/6J) recipients, and the engrafted donor satellite cells (ZsGreen+) were counted via flow cytometry 4 weeks after transplantation. Data are presented as mean ± SEM. Statistical analysis by one-way ANOVA with Dunnett’s correction (n = 4 for vehicle and n = 5 for LPA and NFA treatment groups).These studies used adult (8–16 weeks of age) male mice.