Fig. 1. Mstn inhibits SC proliferation. (A) Isolated single muscle fibers (n=30) were treated with increasing amounts of Mstn. After 48 hours, the fibers were fixed for immunofluorescence to detect BrdU-positive SCs. Increasing Mstn levels reduced the number of BrdU-positive SCs. (B) In the rescue experiment, single muscle fiber cultures were maintained in FM with 1 µg/ml Mstn either throughout the experiment (FM + 1 µg/ml Mstn) or for the first 24 hours, and then in FM without Mstn (Rescue) for a further 24 hours. Control muscle fibers were maintained in FM media throughout the experiment. The activated SCs were identified by BrdU incorporation and immunofluorescence. Whereas addition of Mstn inhibits the activation of SCs, removal of Mstn from the media rescues the SCs from the inhibitory effects of Mstn (^***P<0.001). (C) Single muscle fibers (n=30) were isolated from muscle and incubated in media conducive to the migration of satellite cells in the presence of increasing concentrations of Mstn (^***P<0.001). The average number of migrated satellite cells at varying Mstn concentrations is shown. (D, i) Example of a typical myofiber with BrdU-positive nuclei. (D, ii) The same myofiber with DAPI-stained nuclei.