Collapsed individual myofibers induce MPC proliferation. (A) Representative X-gal staining of intact and collapsed myofibers isolated from the EDL muscles of Myf5-nLacZ mice after 0 and 6 days of culture. Arrows indicate Myf5+ cells. (B) Representative photomicrographs of intact and collapsed myofibers cultured for 0 and 6 days and immunostained with a Pax7-specific antibody (red) and stained with fluorochrome-conjugated phalloidin and DAPI to reveal actin filaments (F-actin, green) and nuclei (blue), respectively. The arrows indicate MPCs (Pax7+ cells). (C) Scatter dot plot showing the number of Pax7+ cells per intact or collapsed myofiber after 6 days of culture. Myofibers from the EDL muscles of three adult WT mice were immunolabeled with a Pax7-specific antibody. Nuclei were stained with DAPI (n = 0 to 43 myofibers). **P = 0.0024 versus intact myofibers. (D) Percentage of the distribution of the number of Pax7+ cells per intact and collapsed myofiber after 6 days of culture. (E) Bar graphs representing the proportion of MPCs in various differentiation states for intact or collapsed myofibers after 0 and 6 days of culture. Following immunostaining of MPCs with Pax7 and MyoD, the differentiation states were defined in terms of combinations of positive staining: quiescent (Pax7+MyoD−), proliferative (Pax7+MyoD+), and differentiating (Pax7−MyoD+). The results are from three independent experiments. Results are expressed as means ± SEM. **P < 0.01; ***P < 0.0001 versus intact and freshly isolated myofibers.