Mice were killed by cervical dislocation and myofibres were inhibitor Pazopanib isolated as previously described. Briefly, extensor digitorum longus muscles were dissected from the hindlimbs and digested in collagenase D for one hour at 37 C. Individual fibres were plated onto glass bottom dishes coated in 10% matrigel, and ei ther fixed immediately in 2% paraformaldehyde or cultured in plating medium for up to several days in Dulbeccos Minimum Essential Medium, 10% horse serum, 0. 5% chick embryo extract with streptomycin and penicillin at 37 C in 5% CO2. In order to de termine whether satellite cells had entered into the cell cycle, myofibres were labelled with 10 uM Bromo deoxy Uridine at the time of plating and harvested after 24 hours in culture.

In order to generate primary Inhibitors,Modulators,Libraries myoblast cultures, myofi bers were washed from the plates after three days of cul ture and the medium was switched to growth medium containing DMEM, 10% HS, 20% fetal bovine serum, 1% CEE, and 2. 5 ngmL basic fibroblast growth factor. Myoblasts were maintained in this medium for up to sev eral days. As cells reached 50 70% confluence, they were passaged after pre plating for 15 minutes on matrigel coated dishes to remove fibroblasts, and plated on fresh matrigel coated dishes. The purity of the myoblast cultures was estimated by desmin staining to be 95%. In order to maintain the characteristics of the cells, all experiments were carried out on myoblasts that had undergone 4 7 passages. For experiments where cells were differentiated, cells were plated on matrigel coated dishes and grown until 50% confluent.

At that time, GM was exchanged for dif ferentiation medium containing DMEM, 2% HS, 10% Inhibitors,Modulators,Libraries FBS, 0. 5% CEE, and antibiotics. Cells were differen tiated for 48 hours unless otherwise stated, Inhibitors,Modulators,Libraries harvested and analysed. At the time of harvest, primary myoblasts were fixed in 2% PFA for 15 minutes and washed several times in phosphate buffered saline and prepared for immunostaining. Adenoviral preparation All adenoviral and corresponding control vectors were obtained from MP Biomedicals. Full length mouse DUOXA1 was cloned into the BglII site of Inhibitors,Modulators,Libraries the CMV5 IRES EGFP AdenoVatorTM vector to create CMV5 DUOXA1 Inhibitors,Modulators,Libraries IRES EGFP and sequencing was per formed. The final adenoviral vector was created by homologous recombination of the aforementioned vec tor with AdEasy, and virus was generated and amplified in 293T cells. Viral purification was Y-27632 FDA achieved using an Adeno X Virus Purification Kit. CMV5 IRES EGFP containing virus was used as the corresponding control. Viral infection Primary myoblasts were plated and maintained in growth medium until they reached 50 60% confluence. At this time, cultures were infected with either CMV5 DUOXA1 IRES EGFP or CMV5 IRES EGFP containing viruses.