recombinant mouse thbs4 protein  (R&D Systems)

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Thbs3 reduces surface integrin levels and destabilizes the sarcolemma. a Representative Western blots of sarcolemma protein extracts from hearts of tTA control, Thbs3 DTG, and Thbs4 DTG mice, assayed for integrin proteins and β-dystroglycan. Laminin2α2 and Cacna1c serve as loading controls. Quantitation is shown in Supplementary Figure . The red box shows integrin proteins specifically reduced on the sarcolemma by Thbs3. b Western blots for input of microsomal protein extracts before immunoprecipitation for Thbs3 and β1D integrin and Cacna1c from NRVMs infected with Adβgal or AdThbs3 (left panel). The microsomal fraction was then immunoprecipitated (IP) for β1 integrin and blotted for the indicated proteins (right panel). Mouse IgG was used as a control for the IP. c Regimen of Evans blue dye (EBD) and isoproterenol (Iso, 300 mg/kg) injection to measure membrane permeability in the hearts of mice. d Representative histological heart sections from 3 tTA control and 3 Thbs3 DTG mice, imaged for WGA (green fluorescence) and leakage of EBD into the cardiomyocytes by red fluorescence subjected to the experimental regimen shown in c . Scale bars are 1 mm. e Regimen of Evans blue dye (EBD) injection after TAC surgery to measure membrane permeability in adult mice. Mice were sacrificed (Sac) 24 h after EBD injection, 1 week after TAC surgery. f , g Representative cardiac histological images of tTA control and Thbs3 DTG hearts 1 week after TAC surgery stained with WGA (green) and intracellular leakage of EBD (red) according to the experimental scheme shown in e , while g shows quantitation of EBD positive cells in the hearts of these mice. Scale bars are 100 μm. * P < 0.05 versus tTA control sham; two-tailed students T -test. Data are represented as percentage EBD positive cells (≥3000 cells from ≥8 animals). Error bars are +/− standard error of the mean and number of mice used is shown in the graph as individual data points

Article Snippet: The denaturation reaction was carried out at room temperature for 40 min. Denaturated luciferase was diluted 125 fold in refolding buffer (25 mM Hepes, pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 1 mM ATP) supplemented with 2 μM BSA or 2 μM of the indicated chaperone protein and incubated at room temperature for 60 min (recombinant heat shock protein 70 (Enzo Life Sciences, #ADI-NSP-555-D), recombinant Thbs4 (R&D Systems #7860-TH-050), recombinant Nell2 (Aviva Systems Biology #OPPB00457), bovine serum albumin (BSA, ThermoFisher Scientific, #BP1600), recombinant Thbs3 (R&D Systems, #8390-TH-050)).

Techniques: Western Blot, Control, Quantitation Assay, Immunoprecipitation, Infection, Injection, Membrane, Permeability, Fluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Integrin overexpression reduces Thbs3-mediated membrane instability and disease. a Schematic of the breeding used to generate combinatorial Thbs3 DTG, α7β1D integrin TG mice. b Representative Western blots for Thbs3 and the indicated integrin proteins from heart sarcolemma protein extracts from tTA, Thbs3 DTG, α7β1D integrin TG and Thbs3 DTG/ α7β1D integrin TG mice. The α1Na + /K + -ATPase served as loading control. c Quantification of EBD positive area from cardiac histological sections after Iso (300 mg/kg) injection in Thbs3 DTG, α7β1D integrin TG, Thbs4 DTG, and Thbs3 DTG/ α7β1D integrin TG mice. d Representative histological images of heart sections from tTA, Thbs3 DTG and Thbs3 DTG/ α7β1D integrin TG mice stained with WGA-FITC (green) after Iso injection (300 mg/kg). EBD is shown as red fluorescence. Scale bars are 300 μm. e Fractional shortening (FS) percentage as determined by echocardiography following 2 weeks of continuous Iso infusion (60 mg/kg/day) or PBS vehicle controls. Number of mice analyzed is shown within each histogram in c , e . * P < 0.05 versus vehicle treated; #<0.05 versus Thbs3 DTG with Iso. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test. Error bars are +/− standard error of the mean

Article Snippet: The denaturation reaction was carried out at room temperature for 40 min. Denaturated luciferase was diluted 125 fold in refolding buffer (25 mM Hepes, pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 1 mM ATP) supplemented with 2 μM BSA or 2 μM of the indicated chaperone protein and incubated at room temperature for 60 min (recombinant heat shock protein 70 (Enzo Life Sciences, #ADI-NSP-555-D), recombinant Thbs4 (R&D Systems #7860-TH-050), recombinant Nell2 (Aviva Systems Biology #OPPB00457), bovine serum albumin (BSA, ThermoFisher Scientific, #BP1600), recombinant Thbs3 (R&D Systems, #8390-TH-050)).

Techniques: Over Expression, Membrane, Western Blot, Control, Injection, Staining, Fluorescence

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Thbs3 enhances secretory pathway activity but reduces membrane integrins. a Time course of intracellular trafficking fluorescence changes in cultured neonatal ventricular cardiomyocytes (NRVMs) infected with a GalNac-T2-RFP baculovirus and the indicated adenoviruses. The data show a quantitative time course of GalNac-T2-RFP recovery in the Golgi network after FRAP to measure ER-to-Golgi vesicular trafficking. * P < 0.05 versus Adβgal infected cells. b Quantitative time course of loss of VSVG-eGFP fluorescence in the Golgi after iFRAP as a measurement for Golgi-to-membrane (Golgi exit) vesicular trafficking. * P < 0.05 versus Adβgal infected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. c Western blots for ECM proteins using heart extracellular matrix extracts from tTA, Thbs3 DTG and Thbs4 DTG mice at baseline or with TAC stimulation. A silver-stained gel is shown with a non-specific (n.s.) band as a loading control. d , e Quantitative time-course of the loss of α5 integrin-GFP fluorescence at the Golgi after iFRAP as a measure of Golgi-to-plasma membrane vesicular trafficking of α5 integrin. The data were generated by iFRAP of α5 integrin-GFP from COS-7 cells d transfected with the plasmids harboring the cDNAs shown or e treated with recombinant Thbs3 or Thbs4 proteins, or bovine serum albumin (BSA) as a control. * P < 0.05 versus Adβgal transfected cells; #<0.05 versus Thbs3 transfected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. Results were summed from four independent experiments and error bars are +/− standard error of the mean

Article Snippet: The denaturation reaction was carried out at room temperature for 40 min. Denaturated luciferase was diluted 125 fold in refolding buffer (25 mM Hepes, pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 1 mM ATP) supplemented with 2 μM BSA or 2 μM of the indicated chaperone protein and incubated at room temperature for 60 min (recombinant heat shock protein 70 (Enzo Life Sciences, #ADI-NSP-555-D), recombinant Thbs4 (R&D Systems #7860-TH-050), recombinant Nell2 (Aviva Systems Biology #OPPB00457), bovine serum albumin (BSA, ThermoFisher Scientific, #BP1600), recombinant Thbs3 (R&D Systems, #8390-TH-050)).

Techniques: Activity Assay, Membrane, Fluorescence, Cell Culture, Infection, Western Blot, Staining, Control, Clinical Proteomics, Generated, Transfection, Recombinant

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( 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 . ( B,C ) Western blot for the expression of Thbs4, ATF6α-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 ATF6α-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 nm DOI: http://dx.doi.org/10.7554/eLife.17589.003

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Labeling, Transmission Assay, Electron Microscopy, Over Expression

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) RelativeThbs4 mRNA levels in human skeletal muscle biopsies from normal, healthy subjects (n = 18) or patients with acute quadriplegic myopathy (AQM; n = 5), juvenile dermatomyositis (JDM; n 21), amyotophic lateral sclerosis (ALS; n = 9), spastic paraplegia (SPG4; n = 4), fascioscapulohumeral muscular dystrophy (FSHD; n = 14), Emery Dreifuss muscular dystrophy (EDMD; n = 7), Becker muscular dystrophy (BMD; n 5), Duchenne muscular dystrophy (DMD; n = 10), calpain 3 mutation (LGMD2A; n = 10), dysferlin (LGMD2B; n = 10), FKRP (Homozygous for a missense mutation Fukutin-related protein; n = 7). *p<0.05 vs. Healthy by Student’s t test. ( B ) Relative protein levels for immunoblots shown in Figure 1B; ( C ) Relative protein levels for ; and ( D ) relative protein levels for . An additional set of immunoblots was included to obtain an n = 4 per genotype. Relative Thbs4 protein levels were determined by quantifying both bands observed for Thbs4. *p<0.05 vs WT; #p<0.05 vs Thbs4 Tg by one-way ANOVA with post hoc Tukey’s test. DOI: http://dx.doi.org/10.7554/eLife.17589.004

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Western Blot

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Glycosylation pattern of Thbs4 protein from quadriceps protein extracts of Thbs4-Tg mice treated with enzymes endoglycosidase H (Endo H) and peptide N -glycosidase F (PNGase F) and compared with a total deglycosylation enzyme mix (Deglycos.). Gapdh is presented as processing and loading control (n = 4 biological replicates). Endo H cleaves high mannose residues and hybrid oligosaccharides present on proteins in the ER while PNGase cleaves both these and more complex oligosaccharides that result from processing in the Golgi. Here, both enzymes equally reduced the apparent size of Thbs4, consistent with high mannose residues typical for proteins in the ER lumen, and proteins that are transitioning to the Golgi to be secreted. ( B ) Representative Western blot analysis for Thbs4 from extracellular matrix (ECM) enriched protein fractions from WT and Thbs4-Tg quadriceps. Laminin is presented as ECM protein control; Gapdh as cytosolic contamination control (n = 3 biological replicates). The data show that whileoverexpressed Thbs4 in non-diseased muscle cannot be readily observed by immunohistochemistry from muscle sections, a more sensitive analysis with ECM-specific western blotting does show some Thbs4 protein outside the myofibers. ( C ) Micrographs representing co-immunofluorescent labeling of Thbs4 (green) with periostin (red) on cryo-embedded Quad of WT, Thbs4-Tg and Sgcd -/- mice at three months of age (scale bar = 50 μm). Nuclei are visualized in blue. Arrowheads indicate co-localization of Thbs4 with periostin in the extracellular milieu during disease; the star marks a myofiber with both intra- and extracellular Thbs4 labeling. Representative images of 4 mice per genotype are shown. ( D ) Quantitation of sub-sarcolemmal vesicular expansion relative to the length of the sarcolemma on transmission electron microscopy images of WT and Thbs4-Tg myofibers from quadriceps at approximately three months of age (n = 22 and 17 myofibers, respectively, from 2 mice per genotype). Data are presented as mean ± SEM; *p<0.0001 vs WT by Student’s t test. DOI: http://dx.doi.org/10.7554/eLife.17589.005

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Glycoproteomics, Control, Western Blot, Immunohistochemistry, Labeling, Quantitation Assay, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Representative images of C2C12 myoblasts cultured with 1 μg/ml Alexa-488 conjugated recombinant Thbs4 (rThbs4-488) or BSA-488 control for the indicated periods. Bright green vesicles containing internalized rThbs4-488, but not BSA-488 were detected in the cytoplasm of C2C12 myoblasts. Cells were counterstained with phalloidin-568 (red); nuclei are visualized in blue. Scale bar = 10 μm. ( B ) Representative images of C2C12 myoblasts cultured with 1 μg/ml rThbs4-488 or BSA-488 control for 24 hr showing partial co-localization of Thbs4 with a late endosome marker (Rab7) but not BSA-488 control. Phalloidin (red) was used to visualize cells; nuclei are visualized in blue. Scale bar = 10 μm. ( C ) Representative images of C2C12 myotubes cultured with 1 μg/ml rThbs4-488 or BSA-488 control for 24 hr. The regions marked by dotted boxes are shown on the right in higher magnification. Bright green vesicles containing internalized rThbs4-488, but not BSA-488 were detected in the cytoplasm of C2C12 myotubes after recombinant Thbs4-488 was added to the media for 24 hr. Phalloidin (red) was used to visualize cells; nuclei are visualized in blue. Scale bar = 25 μm. ( D , E ) Representative immunoblots and semi-quantitative analysis of rThbs4 internalization after addition of 1 μg/ml biotin labeled rThbs4 to the cultured media of C2C12 myotubes for indicated times. n = 3 experiments for all experiments presented in this figure. *p<0.05 vs untreated myotubes by Student’s t test. DOI: http://dx.doi.org/10.7554/eLife.17589.006

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Cell Culture, Recombinant, Control, Marker, Western Blot, Labeling

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Masson’s trichrome stained sections of quadriceps (Quad.) and diaphragm (Diaph.) from WT, Thbs4-Tg, Sgcd -/- and Sgcd -/- Thbs4-Tg mice at 3 mo of age (Scale bar = 100 μm). Blue staining is fibrosis. ( B ) Quantitation of serum CK levels (units/liter) in indicated genotypes at 3 mo of age. *p<0.05 vs WT; #p<0.05 vs Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. N = 8 mice for WT, Thbs4 Tg and Sgcd -/- Thbs4 Tg and n = 7 mice for Sgcd -/- . The legend in this panel also refers to panels C – F . ( C ) Percentage of myofibers with centrally located nuclei in Quad and Diaph from H&E-stained histological sections at 3 mo of age. Representative images are shown in . *p<0.05 vs WT; #p<0.05 vs Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. ( D ) Interstitial fibrosis analyzed in trichrome stained histological sections from Quad, gastrocnemius (Gas) and Diaph at three months of age. *p<0.05 vs WT; #p<0.05 vs Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. N = 6 mice for WT and Thbs4 Tg and n = 8 mice for Sgcd -/- and Sgcd -/- Thbs4 Tg in panel A , C and D . ( E ) Time to fatigue in seconds with forced downhill treadmill running at 3 mo of age in the indicated genotypes of mice. *p<0.05 vs WT; #p<0.05 vs Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. N = 6 mice per genotype. ( F , G ) Quantitation of total Evan’s blue dye (EBD) positive fibers and representative immunofluorescent images of EBD uptake (red) in Quad of three month-old mice. Membranes of myofibers are shown in green. *p<0.05 vs WT; #p<0.05 vs Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. N = 5 mice per genotype in panel F . Scale bar = 100 μm. ( H ) Transmission electron microscopy of quadriceps muscle in Sgcd -/- Thbs4 Tg mice compared to Sgcd -/- mice at 3 mo of age. The arrowheads show subsarcolemmal vesicular expansion due to the Thbs4 Tg. Representative images of 2 mice per genotype are shown. Scale bar = 500 nm. All data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.17589.007

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Staining, Quantitation Assay, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Masson’s trichrome-stained histological sections of quadriceps (Quad.) and diaphragm (Diaph.) from WT, Thbs4-Tg, Sgcd -/- and Sgcd -/- Thbs4-Tg mice at one year of age. Blue color represents fibrosis. Representative images of 6 mice per genotype are shown. Scale bars = 100 μm. ( B , C ) Quantitation of serum creatine kinase (CK) levels (units/liter) and time to fatigue in seconds with forced downhill treadmill running for the indicated genotypes at one year of age. For panel B , n = 9 mice for WT, n = 8 mice for Thbs4 Tg, n = 6 mice for Sgcd -/- and n = 7 mice for Sgcd -/- Thbs4 Tg; and for panel C , n = 5 mice for WT and Thbs4 Tg, and n = 6 mice for Sgcd -/- and Sgcd -/- Thbs4 Tg. Data are presented as mean ± SEM; *p<0.05 vs WT; # p<0.05 vs. Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. ( D ) Masson’s trichrome stained histological sections of quadriceps (Quad.) and diaphragm (Diaph.) from WT, Thbs4-Tg, mdx and mdx Thbs4-Tg mice at one year of age. Blue color represents fibrosis. Representative images of 6 mice per genotype are shown. Scale bars = 100 μm. ( E , F ) Quantitation of serum CK levels (units/liter) and time to fatigue in seconds with forced downhill treadmill running for the indicated genotypes at one year of age. For panel E , n = 9 mice for WT, n = 8 mice for Thbs4 Tg, n = 10 mice for mdx and n = 6 mice for mdx Thbs4 Tg. For panel F , n = 5 mice for WT and Thbs4 Tg, n = 7 mice for mdx and n = 6 mice for mdx Thbs4 Tg. Data are presented as mean ± SEM; *p<0.05 vs WT; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. DOI: http://dx.doi.org/10.7554/eLife.17589.008

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Staining, Quantitation Assay

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Masson’s trichrome histological staining of mdx and mdx Thbs4 Tg quadriceps at three months of age. Blue areas represent fibrosis. Representative images of 6 mice for mdx and 7 mice for mdx Thbs4 Tg are shown. Scale bars = 100 μm. ( B ) Percentage of myofibers with centrally located nuclei in histological sections from quadriceps (Quad) and diaphragm (Diaph) in the indicated groups of mice at three months of age. Representative images are shown in . N = 6 mice for WT and Thbs4 Tg, n = 7 mice for mdx and n = 8 mdx Thbs4 Tg. *p<0.05 vs WT; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. ( C ) Interstitial fibrosis analyzed in Quad and Diaph from histological sections at three months of age in the indicated groups of mice. N = 7 mice for WT and mdx Thbs4 Tg, n = 6 mice for Thbs4 Tg and mdx . *p<0.05 vs WT; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. ( D ) Quantitation of serum CK levels (units/liter) in the indicated genotypes of mice at three months of age. n = 7 mice for mdx and n = 6 mdx Thbs4 Tg. *p<0.05 vs WT; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. ( E ) Time to fatigue in seconds with forced downhill treadmill running at three months of age in the indicated genotypes of mice. N = 6 for indicated genotypes. *p<0.05 vs WT; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. ( F , G ) Representative immunofluorescent images and quantitation of total Evan’s blue dye (EBD) uptake and positive fibers (red) in Quad in three month-old mice subjected to downhill running for 30 min during a 24 hr period of time when EBD is circulating in the mouse. Membranes of myofibers are shown in green. Scale bars = 75 μm. n = 6 for indicated genotypes. *p<0.05 vs WT; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. ( H ) Transmission electron microscopy of quadriceps showing preservation of muscle architecture with a dramatic sub-sarcolemmal accumulation of ER and vesicles (arrows) in mdx Thbs4-Tg mice, compared with noticeable disease and few vesicles in mdx . Representative images of 2 mice per genotype are shown. Scale bar: 500 nm. DOI: http://dx.doi.org/10.7554/eLife.17589.009

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Staining, Quantitation Assay, Transmission Assay, Electron Microscopy, Preserving

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A , B ) representative images and higher magnification inset from H&E-stained histology from quadriceps at three months of age from the indicated genotypes (Scale bar = 100 μm and 50 μm, respectively). Note the significant reduction in myofiber degeneration-regeneration cycles marked by reduced centrally located nuclei upon overexpression of Thbs4 in both Sgcd -/- and mdx quadriceps. Quantification is shown in and . DOI: http://dx.doi.org/10.7554/eLife.17589.010

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Staining, Over Expression

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Separate three day-old Sgcd -/- mouse pups were injected in the gastrocnemius (Gastroc.) with 1E10 viral particles of either adeno-associated virus 9 (AAV9)-Thbs4 or AAV9-eGFP control. The gastroc. was then harvested at six weeks of age for further analysis. ( B ) Representative Western blots showing robust overexpression of eGFP or Thbs4 (Gapdh as loading control, n = 3 biological replicates) in Sgcd -/- gastrocnemius treated with AAV9-eGFP or AAV9-Thbs4, respectively. ( C ) Representative H&E and Masson’s trichrome-stained histological sections from Sgcd -/- gastrocnemius (Gastroc.) treated with control AAV9-eGFP or experimental AAV9-Thbs4 at six weeks of age (separate mice). Representative images of 5 mice per genotype are shown. Scale bar = 100 μm. ( D , E ) Percent myofiber central nucleation and fibrosis from histological sections of the gastrocnemius taken from Sgcd -/- mice six weeks after AAV9-Thbs4 or control AAV9-eGFP treatment. n = 5 mice per treatment group. Data are represented as mean ± SEM; *p<0.05 vs. eGFP by Student’s t test. DOI: http://dx.doi.org/10.7554/eLife.17589.011

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Injection, Virus, Control, Western Blot, Over Expression, Staining

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Masson’s trichrome stained histological sections of Quad and Diaph at three months (mo) of age in Thbs4 -/- , Sgcd -/- and Sgcd -/- Thbs4 -/- mice. Representative images of 6 mice for WT and Thbs4 Tg mice and 8 mice for Sgcd -/- and Sgcd -/- Thbs4 Tg. Scale bar = 100 μm. ( B ) Quantitation of serum CK (units/liter) in the indicated genotypes shown in the legend, at 3 mo of age. *p<0.05 vs. WT; # p<0.05 vs Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. N = 5 mice for WT and n = 6 mice for Thbs4 -/- , Sgcd -/- and Sgcd -/- Thbs4 -/- . ( C ) Time to fatigue in seconds with forced downhill treadmill running in mice at three months of age. *p<0.05 vs. WT; #p<0.05 vs. Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. N = 5 mice per genotype. The legend above panels B and C applies to the remainder of the figure. ( D , E ) Representative immunofluorescent images of EBD (red fluorescence) uptake and quantitation in Quad histological sections from three month-old mice. Membranes of myofibers are shown in green. *p<0.05 vs WT; #p<0.05 vs. Sgcd -/- by one-way ANOVA with post hoc Tukey’s test. N = 5 and 6 mice for Sgcd -/- and Sgcd -/- Thbs4 -/- , respectively. Scale bar = 40 μm. ( F , G ) Time to fatigue in seconds with forced downhill treadmill running and quantitation of serum CK levels in WT and Thbs4 -/- mice at the indicated ages; abbreviations, y = year. n = 6 mice per genotype per age for panel F . For panel G , n = 7 mice per genotype at six weeks of age; n = 5 WT and 6 Thbs4 -/- mice at three months of age, n = 5 mice per genotype at six months of age and n = 9 WT and 8 Thbs4 -/- mice at one year of age. *p<0.05 vs WT at the same age by Student’s t test. ( H ) H&E histological staining (upper) and transmission electron microscopy (lower) of tissue pathology in Quad at one year of age in WT and Thbs4 -/- mice. The H&E scale bar = 25 μm. The electron microscopy scale bar = 2 μm. The arrows show myofibers with central nucleation due to loss of the Thbs4 gene. Representative images of 6 mice per genotype for H&E staining and 2 mice per genotype for electron microscopy. ( I ) Percentage of myofibers with centrally located nuclei in Thbs4 -/- compared to WT Quad at one year of age. *p<0.001 vs WT by Student’s t test. n = 6 mice per genotype. ( J ) Masson’s trichrome stained (upper) and EBD uptake (lower) histological images in WT and Thbs4 -/- mice in the Diaph at one year of age. The EBD images show membranes in green and fibers with EBD uptake produce red fluorescence. Representative images of 6 mice per genotype studied. Scale bars = 50 μm. All data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.17589.012

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Staining, Quantitation Assay, Fluorescence, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Masson’s trichrome-stained histological sections of quadriceps (Quad) and diaphragm (Diaph) from mdx and mdx Thbs4 -/- mice at three months of age. Blue staining shows fibrosis. Representative images of 6 mice per genotype are shown. Scale bars = 100 μm. ( B ) Time to fatigue in seconds with forced downhill treadmill running of the indicated genotypes of mice at three months of age. n = 6 mice per genotype. *p<0.05 vs WT and Thbs4 -/- ; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. ( C ) Quantitation of serum CK (units/liter) levels in the indicated genotypes of mice at three months of age. n = 9 mice for WT and n = 6 mice for Thbs4 -/- , mdx , and mdx Thbs4 -/- . *p<0.05 vs WT and Thbs4 -/- ; # p<0.05 vs. mdx by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.17589.013

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Staining, Quantitation Assay

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Schematic representing the first of 3 consecutive lengthening contraction-induced muscle injury cycles using an in situ tibialis anterior (TA) muscle preparation. Briefly, an isometric contraction was performed to determine baseline force generation, followed by 2 consecutive eccentric contractions and finally another isometric contraction (= injury cycle 1). The force deficit shown in panels B and C was calculated between the first and second isometric contraction in between the two lengthening-contractions, which was repeated 3 cycles total. ( B , C ) Reduction in isometric force generation as a percentage of baseline force after each lengthening contraction injury cycle in the indicated genotypes of mice shown in the legend. *p<0.05 vs WT; # p<0.05 vs WT and Thbs4-Tg; § P<0.05 vs Sgcd -/- and Thbs4-Tg by one-way ANOVA with post hoc Tukey’s test. n = 6 mice for WT, Thbs4 Tg and Sgcd -/- Thbs4 Tg and n = 10 mice for Sgcd -/- for panel B . n = 6 mice for WT, n = 10 mice for Sgcd -/- and n = 5 mice for Thbs4 -/- for panel C . ( D ) Representative images before and after laser injury and influx of FM1-43 dye (green fluorescence) in FDB myofibers in the presence of 1.25 mM Ca 2+ isolated from indicated genotypes. The white tag in each image is the position for the laser injury. Scale bars = 10 μm. ( E , F ) Quantitative time course in seconds of FM1-43 fluorescent dye entry in FDB myofibers from the indicated genotypes of mice in the presence of 1.25 mM Ca 2+ . Laser injury occurred at 60 s. n = 6 fibers per animal from 3 animals per genotype for panels D – F ; *p<0.05 vs WT; #p<0.05 vs Sgcd -/- ; § p<0.05 vs Thbs4 -/- by one-way ANOVA with post hoc Tukey’s test. Data points for Sgcd -/- in panel E and F were derived from a single set of experiments. All data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.17589.014

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: In Situ, Fluorescence, Isolation, Derivative Assay

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Passive tibialis anterior (TA) muscle elasticity measured prior to the lengthening contraction protocol in WT (n = 6), Thbs4 Tg (n = 6) and Thbs4 -/- mice (n = 12). *p<0.05 compared WT by Student’s t test. Data are presented as mean ± SEM. ( B ) Specific isometric force generation averaged over 5 contractions at L 0 from in situ TA preparations for the indicated genotypes. n = 6 mice for WT, Thbs4 Tg, and Sgcd -/- Thbs4 Tg, n = 10 mice for Sgcd -/- , n = 5 for Thbs4 -/- and n = 8 for Sgcd -/- Thbs4 -/- . p-value is not significant by one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM. ( C ) Percentage of tendon breaks recorded during the during the lengthening contraction protocol in WT, Thbs4 -/- and Sgcd -/- Thbs4 -/- mice. Tendon breaks were assessed by complete physical rupture of the tendon at the muscle aponeurosis. n = 6 for WT, n = 10 for Sgcd -/- , and n = 12 for Thbs4 -/- and for Sgcd -/- Thbs4 -/- . DOI: http://dx.doi.org/10.7554/eLife.17589.015

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: In Situ

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A – D ) Time course of GalNac-T2-RFP or VSVG-eGFP fluorescence changes in cultured neonatal ventricular myocytes infected with the indicated adenoviruses to overexpress Thbs4 (maroon line, n = 18), a dominant negative (dn) ATF6α (turquoise line, n = 8) or in combination with Thbs4 (purple line, n = 13), a constitutively nuclear (cn) ATF6α (green line, n = 12), or βgal expressing control (black line, n = 14). Change in fluorescence was after FRAP or iFRAP to measure ER-to-Golgi or Golgi to the membrane trafficking, respectively. ( E , G , I ) Quantitative time course of GalNac-T2-RFP recovery in the Golgi network after FRAP to measure ER to Golgi vesicular trafficking in primary neonatal rat ventricular myocytes infected with adenoviral Thbs4 (green line, n = 18), Nell2 (red line, n = 9), the Type III repeat (AdT3R; red line, n = 8) domain of Thbs4, the N-terminal Laminin G (AdLamG, red line, n = 7) domain of Thbs4, or βgal control (blue line, n = 14). Shown above these graphs is a schematic diagram depicting the domain structure of Thbs4, Nell2 and T3R or LamG of Thbs4. All data are represented as mean ± SEM. ( F , H , J ) Quantitative time course of loss of VSVG-eGFP fluorescence in the Golgi after iFRAP as a measurement for Golgi-to-membrane (Golgi exit) vesicular trafficking in primary neonatal rat ventricular myocytes infected with adenoviral Thbs4 (green line, n = 18), Nell2 (red line, n = 8), the Type III repeat (AdT3R; red line, n = 8) domain of Thbs4, the N-terminal Laminin G (AdLamG; red line, n = 7), or βgal control (blue line, n = 14). All data are represented as mean ± SEM. Each cell imaged per experimental condition represents an independent experiment. DOI: http://dx.doi.org/10.7554/eLife.17589.016

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Fluorescence, Cell Culture, Infection, Dominant Negative Mutation, Expressing, Control, Membrane

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Representative images showing the recovery of GalNacT2-RFP fluorescence (red) into the photobleached Golgi network (FRAP; outlined green area) over time in primary neonatal rat ventricular myocytes infected with an adenovirus expressing Thbs4 (AdThbs4) or a βgal control. Recovery of Golgi fluorescence was monitored as a measure for ER-to-Golgi vesicular trafficking. Scale bar = 10 μm. ( B ) Representative images of VSVG-eGFP fluorescence (green) before and after inverse FRAP (iFRAP, outlined area, red) in primary neonatal rat ventricular myocytes. Adenovirus expressing Thbs4 or βgal (control) were used. Reduction in Golgi fluorescence (outlined in yellow) was monitored as a measure for Golgi-to-membrane vesicular trafficking. Scale bar = 25 μm. ( C ) Western blots from quadriceps muscle of WT and Thbs4 Tg mice for vesicular markers associated with ER to Golgi trafficking: Sar1, Rab24 and Rab6, and vesicular markers associated with Golgi-to-membrane trafficking (secretory vesicles): Rab3 and Rab8 (n = 3 biological replicates). Gapdh is presented as processing and loading control. DOI: http://dx.doi.org/10.7554/eLife.17589.017

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Fluorescence, Infection, Expressing, Control, Membrane, Western Blot

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Immunofluoresence (green) detection of δ-, α-, β-, and γ-sarcoclycan (SGC), β-dystroglycan (β-DG), utrophin (Utro.), dystrophin (Dystro.) and β1D-integrin in littermates of three month-old WT, Thbs4-Tg, Sgcd -/- and Sgcd -/- Thbs4-Tg quadriceps. Representative images of 4 mice per genotype are shown. Scale bar = 25 μm. ( B ) Representative Western blots of sarcolemmal protein extracts (upper) or total cytoplasmic protein extracts (lower) from the quadriceps of the indicated groups of mice for the indicated proteins (n = 4–5 biological replicates). Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5- integrin. The red boxes show increased protein levels. Also see for replicates. ( C ) Representative immunoblotting for structural components of the DGC- and integrin-associated protein complexes in sarcolemmal preparations from Thbs4 -/- and WT quadriceps at four months of age (n = 4 biological replicates). The burgundy-boxed areas show reduced protein levels. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls for sarcolemmal protein extracts; Gapdh was used as loading control for total cell protein extracts. DOI: http://dx.doi.org/10.7554/eLife.17589.018

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Western Blot, Staining, Control

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A , B ) Representative Western blots of sarcolemmal protein extracts from the quadriceps of the indicated groups of mice at three months of age (n = 4–5 biological replicates). Sgcd -/- Tg and mdx Tg indicate Sgcd -/- and mdx with skeletal muscle specific Thbs4 overexpression, respectively. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls. Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-itg (integrin). ( C ) Immunoblots for β1D- and α7-Integrin (Itg), β-dystroglycan (DG) and Thbs4 (Flag) from neonatal rat ventricular myocyte extracts immunoprecipitated with a Flag antibody (Thbs4). Adβgal was used as a control infection (n = 3 biological replicates). An adenovirus expressing a Flag-tagged Thbs4 protein was used to achieve high level of this protein to identify the interaction. ( D , E ) Representative Western blots for Thbs4, α5- and β1D-integrin (itg) from intracellular vesicular isolates from WT and Thbs4 Tg quadriceps ( D ) or WT and Sgcd -/- quadriceps ( E ) that were immunoprecipitated with an antibody raised against the cytoplasmic domain of β1D-integrin (n = 3 biological replicates), showing that Thbs4 and α5-integrin localize to β1D-integrin-positive intracellular vesicles. α-tubulin and Gapdh are presented as loading control. DOI: http://dx.doi.org/10.7554/eLife.17589.019

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Western Blot, Over Expression, Staining, Immunoprecipitation, Control, Infection, Expressing

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Schematic diagram of the transgene (Tg) used to overexpress ATF6α in skeletal muscle. ( B ) Western blot analysis for Thbs4, ATF6α, BiP, PDI and calreticulin (Calret.) expression in quadriceps (Quad) from WT, Thbs4 Tg and ATF6α Tg mice at six weeks of age. Gapdh is a processing and loading control (n = 6 biological replicates). ( C ) Masson’s trichrome-stained histological sections from Quad of WT and ATF6α-Tg littermates at six weeks of age. Representative images of 5 mice per genotype are shown. Scale bar = 100 μm. ( D ) Transmission electron micrographs in Quad from WT and ATF6α-Tg mice at six weeks of age. The white arrows show dramatic expansion of ER and associated vesicles throughout the cell and especially in the sub-sarcolemmal region. Representative images of 2 mice per genotype are shown. Scale bar = 2 μm. ( E ) Masson’s trichrome stained histological sections of Quad from Sgcd -/- and Sgcd -/- ATF6α Tg mice at six weeks of age. Representative images of 5 mice per genotype are shown. Scale bar = 100 μm. ( F ) Quantitation of serum CK levels (units/liter) in the indicated genotypes of mice shown in the legend below the graph at six weeks of age. n = 10 mice for WT and n = 8 mice for the remaining genotypes. *p<0.05 versus WT by one-way ANOVA with post hoc Tukey’s test. ( G , H ) Histological analysis of the Quad showing percentage of myofibers with centrally located nuclei (n = 5 mice for WT and ATF6α Tg, and n = 6 mice for Sgcd -/- and Sgcd -/- ATF6α Tg) and interstitial fibrosis (n = 5 mice per genotype) at six weeks of age in WT, ATF6α Tg, Sgcd -/- and Sgcd -/- ATF6α Tg mice. *p<0.05 versus WT by one-way ANOVA with post hoc Tukey’s test. ( I ) Time to fatigue in seconds with forced downhill treadmill running in the indicated genotypes of mice shown in the legend. n = 6 mice per genotype. *p<0.05 versus WT by one-way ANOVA with post hoc Tukey’s test. ( J ) Representative immunofluorescent images of EBD (red) uptake in myofibers in the Quad of six week-old mice of the indicated genotypes. Membranes of myofibers are shown in green. Scale bars = 75 μm. Percent EBD-positive myofibers is indicated. Six mice per genotype were analyzed for EDB uptake. ( K ) Western for structural components of the DGC and integrin-associated protein complexes in sarcolemmal protein preparations from Quad of WT, ATF6α Tg, Sgcd -/- and Sgcd -/- ATF6α Tg littermates at six weeks of age. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls (n = 3 biological replicates). Abbreviations: Utro, utrophin; Dystro, dystrophin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-Itg (integrin). All data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.17589.020

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Staining, Transmission Assay, Quantitation Assay

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: An additional set of immunoblots was included to obtain an n = 6 per genotype. *p<0.05 vs WT; #p<0.05 vs Thbs4 Tg by one-way ANOVA with post hoc Tukey’s test. DOI: http://dx.doi.org/10.7554/eLife.17589.021

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Western Blot

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Schematic of the experimental protocol used. Briefly, purified adenoviral (Ad) constructs encoding Flag-tagged full-length Thbs4 (AdThbs4), Flag-tagged Thbs4 calcium-binding mutant (AdThbs4-mCa 2+ , which induces ER-stress but isretained in the ER) or βgal control were injected into the gastrocnemius (Gastroc.) muscles of postnatal day one rat pups, followed by a second ‘boost’ injection 48 hr later (10 8 viral particles per injection). Muscle tissue was harvested at postnatal day eight, cryo-sectioned and co-immunostained for Thbs4 and β1-integrin (Itg). ( B ) Immunofluorescence detection of Thbs4 and Thbs4-mCa 2+ (both Flag tagged; green) and β1-integrin (red) from muscles infected with indicated Thbs4 constructs or βgal control. Representative images of 3 muscles per condition are shown. Scale bar = 10 μm. ( C ) Quantitative analysis of sarcolemma localized β1-integrin positive immunofluorescence. Data are presented as % β1-integrin positive area from total muscle area infected with the indicated Thbs4 constructs (Flag-positive) or from total muscle area in the case of βgal control. N = 3 per treatment condition. *p<0.05 vs. Adβgal and AdThbs4-mCa 2+ by Student’s t test. DOI: http://dx.doi.org/10.7554/eLife.17589.023

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Purification, Construct, Binding Assay, Mutagenesis, Control, Injection, Muscles, Immunofluorescence, Infection

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Survival of the Drosophila lines shown over a period of 40 days. p<0.001 for Drosophila line 840 lacking the δ-sarcoglycan homologue gene (Sgcd 840 ) versus Sgcd 840 ;mThbs4 line that express the mouse Thbs4 protein in muscle. N = 358 for WT; 163 for mThbs4; 395 for Sgcd 840 ; 300 for Sgcd 840 ;mThbs4. Statistical analysis performed with log rank, Mantel-Cox test. ( B ) Physical performance with a negative geotaxis assay of WT (n = 208), mThbs4 (n = 190), Sgcd 840 (n = 203), and Sgcd 840 ;mThbs4 Drosophila (n = 205) at the indicated ages. All data are represented as mean ± SEM. *p<0.05 vs WT and mThbs4; # p<0.05 vsSgcd 840 by one-way ANOVA with post hoc Tukey’s test. ( C ) Representative H&E stained histological sections of the dorsal median indirect flight muscles at 30 days of age in the indicated genotypes of Drosophila . Representative images of 14 Drosophila per genotype studied. The arrow shows a prominent area of muscle rupture in the Sgcd 840 MD Drosophila line. Scale bar = 200 μm. ( D ) Immunohistochemistry showing increased intracellular mThbs4 and calreticulin (Calret.; both green) in longitudinal sections of the dorsal median indirect flight muscle of 30 day-old mThbs4 and Sgcd 840 ; mThbs4 expressing flies compared to WT and Sgcd 840 lines. Membranes are shown in yellow, nuclei in blue. Representative images of 14 Drosophila per genotype studied. Scale bars = 10 μm. ( E ) Immunofluorescence detection of fly βPS integrin (green) in cross-sections the dorsal median indirect flight muscle of 30 day-old Drosophila of the indicated genotypes. Representative images of 10 Drosophila per genotype studied. Scale bars = 10 μm. ( F ) Representative Western blot of mThbs4 and ER-stress proteins BiP, PDI, calreticulin (Calret.), as well as βPS integrin in 15 day-old WT, mThbs4, Sgcd 840 , and Sgcd 840 ;mThbs4 lines (n = 4 biological replicates). β actin was used as loading control. The red boxes show upregulation of βPS integrin with muscle specific mThbs4 overexpression in the WT and Sgcd 840 mutant lines. ( G ) Transmission electron microscopy of the dorsal median indirect flight muscle of WT, mThbs4, Sgcd 840 , and Sgcd 840 ;mThbs4 Drosophila . Arrows indicate the characteristic sarcomeric tears and disorganization present in the Sgcd 840 line. Representative images of 4 Drosophila studied for the WT, mThbs4, and Sgcd 840 ;mThbs4 lines, and 5 Drosophila for the Sgcd 840 line. Scale bar = 2 μm. ( H ) Immunogold-transmission electron microscopy of mThbs4 in longitudinal section of the dorsal median indirect flight muscle of mThbs4 Drosophila . The arrows indicate mThbs4 (6 nm gold particles) localized to intracellular vesicles. Representative images of 4 Drosophila are shown. Scale bar = 50 nm. DOI: http://dx.doi.org/10.7554/eLife.17589.024

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Staining, Muscles, Immunohistochemistry, Expressing, Immunofluorescence, Western Blot, Control, Over Expression, Mutagenesis, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: As a matricellular protein, thrombospondin-4 (Thbs4) pentamers are synthesized in the ER lumen and then transported to the Golgi, where after they traverse the secretory pathway to fuse with the plasma membrane for secretion. Thbs4 can then reside within the extracellular matrix (ECM) or be actively endocytosed and returned to the intracellular compartment by a recycling receptor. In addition to its established extracellular functions, combined studies in the heart and skeletal muscle now reveal that while in the ER, Thbs4 can compete with BiP (GRP78) for binding to the ER-resident transcription factor ATF6α, thereby facilitating ATF6α translocation to the Golgi for processing and subsequent shuttling to the nucleus where it regulates expression of ER stress responsive genes that are also part of the unfolded protein response (UPR). ATF6α induction in cardiac and skeletal muscle, or by overexpression in Tg mice (lower panel) causes a dramatic expansion of the ER and post-ER vesicles, as well as increased vesicular trafficking to the membrane. The ability of Thbs4 to induce ATF6α processing and nuclear trafficking also causes this same ER expansion and augmentation of intracellular vesicular trafficking to the membrane. However, Thbs4 uniquely regulates trafficking of selected integrins and dystrophin-associated glycoprotein complexes (DGC) members to the sarcolemma, thereby enhancing the mechanical stability of the myofiber, such as observed in Thbs4 Tg muscle (middle panel). DOI: http://dx.doi.org/10.7554/eLife.17589.026

Article Snippet: Alexa Fluor-488 Microscale Protein Labeling Kit (Life Technologies, Cat#30006) and EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Cat# 21217) were used to label recombinant mouse Thbs4 (rThbs4, R&D Systems, Cat# 7860-TH-050) according to the manufacturer’s instructions.

Techniques: Synthesized, Clinical Proteomics, Membrane, Binding Assay, Translocation Assay, Expressing, Over Expression