We also performed Western analysis on cell lysates generated from the same satellite cells to observe protein levels of FRG1. Levels of FRG1 protein are increased in both proliferating and differentiated cultures of satellite cells from of H-FRG1TG mice. In addition, FRG1 is detected in satellite cell cultures derived from either diaphragm or thigh muscle. For clonal analysis, the tissue-derived satellite cells were plated at a very low density, 1000 cells per 10 cm dish, and allowed to grow for a predefined amount of time. At regular intervals, plates were fixed and nuclei were visualized by staining with methylene blue. We also stained for myosin heavy chain as a control to verify that cells did not prematurely differentiate over the course of the clonal assay, in which bFGF and high serum levels were maintained. After fixation and staining, the total number of cells per clone was determined and binned for comparison in a histogram format. Thigh-derived satellite cells from an 18-week old H-FRG1TG mouse show a marked decrease in average clone size compared to those derived from a wild-type littermate control. A significant fraction of these cells show arrest in a 2- cell clone size skewing the distribution compared to the wild-type thigh-derived satellite cells. This effect may be even more dramatic considering that BI-D1870 single cell clones were not scored in this assay, as we consider single cell clones may potentially be new clones arising from detached satellite cells floating away from the original clone during mitosis. We replicated these observations with an independent satellite cell culture isolated from 20-week old mouse limbs obtained directly from Dr. Rossella Tupler which were comparable to our 18-week old thigh-derived satellite cells at a similar time point. The proliferative defect was not replicated in the diaphragm-derived satellite cells, which show a very similar clone size distribution between the H-FRG1TG and wild-type C57BL/6 littermates. These findings indicate that FRG1 overexpression leads to a muscle-type OTX015 purchase specific defect in proliferation, and correlates with the dystrophic phenotype. To determine whether there is an age-dependent increase in severity of the observed proliferation defect, we isolated and performed a clonal assay on both thigh- and diaphragm-derived satellite cells isolated from 4-week old mice, which appear asymptomatic, to compare to the aforementioned data from more severely symptomatic 18-week old mice. For each of these populations, we scored multiple time points of a clonal assay, to more thoroughly assay the proliferative defect. The clone size distributions of myoblasts from asymptomatic 4-week old mice did not show any significant proliferative defect when compared to their 18-week old counterparts at a similar clone size.