SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi: /nature10980 SUPPLEMENTARY FIGURES Figure S1 Supplementary data from WT TG(-), WT TG(+), mdx TG(-) and mdx TG(+) mice. a, There was no difference in HSP72 expression in muscle samples from young boys with DMD (aged 1-9 years) compared with muscle samples from young adults (aged years) indicating that HSP72 levels are not elevated to a protective level in DMD (n 8). b, Quantified mean data of HSP72 protein expression in diaphragm muscle homogenates. P < 0.01, n 4. c, Representative haematoxylin and eosin (H & E)-stained diaphragm sections. d, Representative Evans blue dye (EBD) visualised sections of diaphragm muscles from mdx TG(-) and mdx TG(+) mice. e, Susceptibility of diaphragm muscle strips and TA muscles from 80 week-old mice to contraction-induced injury measured in vitro and in situ, respectively (n 3). f, Utrophin A and B mrna expression (normalised to cdna content) in diaphragm and TA muscles from mdx TG(-) and mdx TG(+) mice measured by RT-PCR. g, Quantified mean data for collagen infiltration in diaphragm muscle sections (visualised with Van Gieson s stain). Asterisk, P < 0.01, n 5. Scale bar, 200 μm. Data are from 25 week-old mice unless specified otherwise. Data represented as mean ± SEM. 1

2 RESEARCH SUPPLEMENTARY INFORMATION Figure S2 Western blots and RT-PCR data of inflammatory and macrophage markers in diaphragm muscles from mdx TG(-) and mdx TG(+) mice. Representative western blots and quantification of; a, pikk normalised to tikk; and b, pjnk normalised to tjnk. Quantified mrna expression of; c, the inflammatory mediator TNF-α; and d, the macrophage markers CD68 and F4/80, normalised to cdna content. n 7 for all data. Data are from 25 week-old mice. Data are mean ± SEM. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Figure S3 RT-PCR data of inflammatory and macrophage markers in TA muscles from mdx TG(-) and mdx TG(+) mice. Quantified mrna expression of; a, the macrophage markers CD68 (Asterisk, P < 0.001) and F4/80 (Asterisk, P = 0.001) and; b, the inflammatory marker TNF-α (Asterisk, P = 0.008), normalized to cdna content. n 9 for all data. Data are mean ± SEM. Figure S4 SERCA function in diaphragm muscles from WT and mdx mice. a, Maximal SERCA activity in 25 week old mdx diaphragm muscle homogenates (normalised to age matched WT control). B, Top panels: Mean SERCA 1 and 2a protein expression in WT and mdx diaphragm muscles. Asterisk, P < 0.001, n 4. Bottom panels: Representative western blots of SERCA 1, 2a, and GAPDH. (c) Ca 2+ -dependence curves showing SERCA enzyme kinetics (left panel), and mean [Ca 2+ ] 50 (the [Ca 2+ ] required to elicit 50% of maximal enzyme activation; right panel) in diaphragm muscle homogenates from 30 week old WT and mdx mice. Asterisk, P = 0.026, n 4. Data are mean ± SEM. 3

4 RESEARCH SUPPLEMENTARY INFORMATION Figure S5 SERCA function in mdx TG(-) and mdx TG(+) mice. a, Ca 2+ -dependence curves showing SERCA enzyme kinetics (left panel), and mean [Ca 2+ ] 50 (the [Ca 2+ ] required to elicit 50% of maximal enzyme activation; right panel). n 8. b, SERCA 1 and SERCA 2a mrna expression (normalised to 18S) in diaphragm muscles from mdx TG(-) and mdx TG(+) mice measured by RT-PCR, n 7. c, Representative western blots of SERCA 1, SERCA 2a, HSP72 and GAPDH in EDL (top left panel) and soleus (top right panel) muscle homogenates. Bottom panels: quantified mean SERCA 1, SERCA 2a, and HSP72 protein expression from mdx TG(-) and mdx TG(+) mice. Asterisk, P < 0.001, n 4. d, SR Ca 2+ accumulation curves (an indirect measure of SERCA activity) in single fibres from soleus muscles of mdx TG(-) and mdx TG(+) mice. Asterisk, P < 0.01, n 10. Data are mean ± SEM. 4

5 SUPPLEMENTARY INFORMATION RESEARCH Figure S6 HSP72 expression in enriched SR vesicles isolated from mdx TG(-) and mdx TG(+) mice. Enriched SR vesicles were immunoblotted for a, HSP72 protein and b, stained with Coomassie blue to visualise all proteins on the membrane. The predominant band was at ~ kda corresponding to the SERCA isoforms, the primary constituent protein of enriched SR vesicles. Lanes 1, 2, 5, and 6 are SR vesicles from mdx TG(-) mice. Lanes 3, 4, 7 and 8 are SR vesicles from mdx TG(+) mice. Figure S7 Data from BGP-15 treated mdx mice. (a) Quantified mean data of HSP72 protein expression from whole diaphragm muscle homogenates from control and BGP-15 treated (6 hrs post single dose gavage) mdx mice. P < 0.05, n 3. b, Quantified mean data for collagen infiltration in diaphragm muscle sections (visualised with Van Gieson s stain). P < 0.01, n 7. c, Mean latency-to-fall data from WT, mdx control and mdx BGP-15 mice. P < 0.05, n 10. Data are mean ± SEM. 5

6 RESEARCH SUPPLEMENTARY INFORMATION Figure S8 Core body temperature and HSP72 expression during and after heat therapy (HT). a, Core body temperature was measured in unconscious mice via the external auditory meatus (ear canal) using an infrared thermometer (Thermoscan, Braun). n = 13. Mice were subjected to heat therapy every 4 th day from 4 wks until 9 wks of age. b, Representative western blots and quantification of HSP72 and α-tubulin from control and HT (12 hours post single HT bout) mdx whole diaphragm muscle homogenates. Asterisk, P < 0.05, n 3. c, Whole body muscle breakdown, measured by serum creatine kinase (CK) levels. n 7. d, Mean latency-to-fall data from WT, mdx control and mdx heat therapy mice. n 9. e, Specific (normalised) force of diaphragm muscle strips measured in vitro. Asterisk, P < 0.05, n 9. f, Quantified mean data for collagen infiltration in diaphragm muscle sections (visualised with Van Gieson s stain). P < 0.05, n 7. Data are mean ± SEM. 6

7 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 1. Contractile characteristics of mechanically skinned single fibres from EDL and soleus muscles of mdx TG(-) and mdx TG(+) mice. n 10. (n 7 for leak data only) mdx TG(-) mdx TG(+) EDL sp (kn/m 2 ) 167 ± ± 6 pca ± ± 0.03 n H 7.4 ± ± 0.4 % SR Ca 2+ leak 21 ± 4 20 ± 2 SR Ca 2+ capacity (AU) 9.3 ± ± 0.8 Soleus sp o (kn/m 2 ) 167 ± ± 7 pca ± ± 0.01 n H 6.3 ± ± 0.3 % SR Ca 2+ leak 23 ± 3 22 ± 4 SR Ca 2+ capacity (AU) 4.7 ± ±

8 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 2. Contractile properties of TA muscles and diaphragm muscle strips from 10 week old untreated dko and BGP-15-treated dko mice. n 9 dko dko BGP-15 P-value TA P t (mn) 304 ± ± in situ TPT (ms) 15.7 ± ± dp/dt (mn/ms) 56.3 ± ± ½ RT (ms) 16.1 ± ± P o (mn) 1068 ± ± sp o (kn/m 2 ) ± ± Muscle mass (mg) 66.3 ± ± Diaphragm sp t (kn/m 2 ) 20.0 ± ± in vitro TPT (ms) 37.5 ± ± dp/dt (mn/ms) 4.6 ± ± ½ RT (ms) 38.8 ± ± sp o (kn/m 2 ) 71.2 ± ± 3.0 P< dp/dt, maximum rate of force development during a twitch contraction; Po, maximum isometric force; Pt, peak twitch tension; ½ RT, half-relaxation time; spo, specific force (force per CSA); spt, specific twitch force (force per CSA). P-values based on Students t-test. 8