FIG. 5.

The effect of ECM and mechanical stimulation upon the myoblast secretome. C2C12 cells were cultured in proliferation media or allowed to form myotubes in differentiation media culture. Myoblasts or myotubes were treated with 200 μg/mL of ECM degradation products for 18 h, after which the media were replaced with serum-free, ECM-free media and the cells were subjected to mechanical strain. Conditioned media were collected and added to bone marrow-derived macrophage for 18 h and the cells were fixed for immunolabeling. (A) Exposure times were normalized to cytokine stimulated controls. (B, D) The secretome of proliferating myoblasts promotes an iNOS−/Fizz1+ macrophage phenotype, (C) however, differentiated myotubes do not promote the same effect. Treating myotubes with ECM degradation products, however, alters their secretome allowing them to promote a Fizz1+ macrophage phenotype. (E) This response is augmented when ECM-treated myotubes are subjected to mechanical strain. (F) Percentage of iNOS- and Fizz1-positive macrophages were quantified using CellProfiler. (#p < 0.05 for iNOS expression when compared to the untreated control, *p < 0.05 for Fizz1 expression when compared to the untreated control, n = 4, error bars represent standard error of the mean).