force by means of a grip strength meter (Columbus Instruments, USA) [14,15]. An exercise resistance test, consisting of horizontal running for 5 min at 5 m/min, then increasing the speed of 1 m/ min each minute, was performed on week 0 and after four and five weeks of treatment. The total distance run by each mouse until exhaustion was measured [15]. At the end of the 5th week of exercise/treatment the ex vivo experiments were also started. To this aim mice were deeply anesthetized and sacrificed using 1.2 g/ kg urethane (i.p.) in accordance with the Italian Guidelines for the use of laboratory animals, which conform with the European Community Directive published in 1986 (86/609/EEC). Muscle mechanics/electrophysiology Muscle mechanics were conducted on the extensor digitorum longus (EDL) muscle as previously described [6]. EDL muscles were removed and rapidly placed in the recording chamber for the electrophysiological or isometric recordings. In the recording chambers EDL muscles were bathed at 3061uC in the following normal physiological solution: NaCl 148 mM, KCl 4.5 mM, CaCl2 2.0 mM, MgCl2 1.0 mM, NaHCO3 12.0 mM, NaH2PO4 0.44 mM, glucose 5.55 mM, and continuously gassed with 95% O2 and 5% CO2 (pH 7.2–7.4). The mechanical threshold (MT) was determined in EDL muscle fibres by the two microelectrode ‘‘point’’ voltage clamp method, described previously [13,14]. In brief, depolarizing command pulses of variable duration (from 500 to 5 ms at 0.3 Hz) were progressively increased in amplitude from the holding potential (H) of 290 mV until a visible fiber contraction. The mean threshold membrane potential values of individual myofibers (V, in mV) at various pulse durations (t, in ms) allowed the construction of a ‘‘strength-duration’’ curve. The rheobase voltage (R, in mV) was obtained by a non-linear least square algorithm using a previously described equation [14,15]. Protein analyses A human DMD cell line with a deletion of exons 49 and 50 (generously provided by Vincent Mouly, Paris) was seeded in 6 cm dishes in DMEM (Invitrogen), Medium 199 (20%; Sigma-Aldrich), Fetal Calf Serum (20%; Invitrogen), glutamine and penicillin/ streptomycin (Invitrogen) 24 h prior to drug treatment. After three days of drug treatment cells were lysed for 20 min at 4uC in Tris pH6.8 (75 mM), SDS (3.8%), Urea (4 M), glycerol (20%) supplemented with protease inhibitors (1:100; Sigma-Aldrich). Soluble protein was purified at 80006g for 20 min at 4uC and 30 ml was size fractionated by 5% Tris-HCL SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (GE Healthcare). Soluble protein was prepared from muscle samples snap frozen in liquid nitrogen and stored at 280uC until use for biochemical analysis. For western blotting, protein was crushed with a pestle in a liquid nitrogen-cooled mortar, solubilised in 50 volumes of single-section western blot lysis buffer [16], vortexed, briefly homogenised and sonicated, heated to 94uC for 4 minutes and centrifuged for 3 min at 15 0006g to remove insoluble matter. For western blotting, 50 ml soluble protein extract was separated by 5% Tris-HCL SDS-PAGE gel electro- phoresis and transferred onto PVDF (GE Healthcare). Utrophin protein was detected using MANCHO 3 antibody (1:100; kind gift from G.E. Morris, Oswestry, UK) and ECL HRP-conjugated anti- mouse antibody (GE Healthcare). Equal protein loading was corrected by detection of a-actinin (1:200, N-19; Santa Cruz Biotechnology, Inc.) and a HRP-conjugated anti-goat antibody (Sigma). Blots were developed using ECL reagent (GE Health- care), Densitometry was performed using the freely available web version of Image J (rsbweb.nih.gov/ij/). Immunohistochemistry was carried out as previously described [17]. SMT C1100 Reduces mdx Dystrophic Symptoms RNA analyses For quantitative real time RT-PCR SkMC cells were seeded in six well plates at 25000 cells per well in 3 ml of appropriate media and incubated for 24 h prior to dosing. Compounds were dosed in a final concentration of 1% DMSO for 72 h. RNA was extracted from tissue using a QIAGEN RNeasyH Plus kit (Qiagen) and QiaShredder (Qiagen), using the manufacturer’s instructions. The High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems) was used according to manufacturer’s instructions. Real-time PCR was per- formed according to the DDCT method [18]. The 7300 Real-Time PCR System from Applied Biosystems was used for this assay along with the 7300 System SDS software with the SDS Relative Quanti- fication Study Plug in. Data was analysed using the 7300 System SDS software with the SDS Relative Quantification Study Plug in. Blood analyses Measurements of SMT C1100 plasma concentrations were obtained following RO (retro-orbital) blood sampling on day 1, 24 h after oral dosing with SMT C1100 (50 mg/kg) or vehicle only (0.1% PBS-Tween-20/5% DMSO). Further samples were taken at day 15 and day 28 after the start of dosing. Blood was collected with non-heparinized hematocrit tubes into serum microtainer tubes and centrifuged for 12 min at 12,000 rpm at 4uC. Serum was stored at 280uC prior to analysis using the CK (NAC) reagent kit in conjunction with the AU 400 Clinical Chemistry analyser (Olympus UK Ltd). Histological analyses After 28 days of dosing muscle samples were taken for histopathology and processed by Premier Laboratory LLC (Colorado, U.S.A.). Samples were received in 10% neutral buffered formalin, processed into paraffin, and 5 mm sections and stained for Hematoxylin and Eosin (H&E) and Masson’s Trichrome (tibialis anterior, extensor digitorum longus, soleus, and diaphragm). Both the H&E and Masson’s Trichrome stained slides were submitted blind to a Board Certified Veterinary Pathologist at Premier Laboratory LLC and scored for the presence of inflammation and fibrosis. A total of five sections from each muscle were analysed. The muscle fibres were scored according to the following criteria: Inflammation: 0 = none to minimal - No inflammation within the muscle bundles or inter-bundle connective tissue; occasional mononuclear inflammatory cells may be present but no obvious aggregations. 1 = mild - Occasional mononuclear inflammatory cells in the inter-bundle connective tissue with focal aggre- gations of mononuclear inflammatory cells. 2 = moderate - Multiple foci of mononuclear inflamma- tory cell infiltration in the inter-bundle connective tissue; occasional mononuclear inflammatory cells between individual muscle fibres. 3 = severe - Multiple large foci of mononuclear inflamma- tory cell infiltration in the inter-bundle connective tissue extending into the intra-bundle connective tissue with expansion of the inter-bundle and intra-bundle space. Fibrosis: 0 = none to minimal - No fibrosis in the muscle bundles or inter-bundle connective tissue; mild expansion of the inter-bundle connective tissue may be present focally. PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19189