Fig. 4. Mmp13^-/- myoblasts in vitro have impaired migration. (A) A brightfield frame of live cell microscopy with multicolored fiber tracks showing the course of migration over 14 hours in WT and Mmp13^-/- primary satellite cells. A fiber is shown on the left side of the Mmp13^-/- frame. Scale bar is 100 μm. Representative videos are included in the supplement. (B) Average velocity of migrating cells in 2D from live cell experiments were similar in WT cells (N=307) and Mmp13^-/- cells (N=215) at 20% serum and significantly different between WT cells (N=27) and Mmp13^-/- cells (N=35) in 2% serum. Numbers of cells analyzed are shown on each column of the graph. (C) Proliferation rate measured as cells are migrating in 2D obtained from the same experiments as in B, which consisted of cells from WT (N=9) fibers and Mmp13^-/- (N=7) fibers in 20% serum and WT (N=6) fibers and Mmp13^-/- (N=6) fibers in 2% serum were not significantly different between genotypes. (D) Examples of 3D migration of WT and Mmp13^-/- cells showing bottom side of membrane with cells invading the designated substrate (basement membrane extract (BME) or collagen) over the course of 24 hours. Scale bar = 50 μm. (E) 3D migration of Mmp13^-/- cells through BME is impaired compared to WT cells, and almost non-existent through collagen by cells of both genotypes. 3 images per membrane (N=3 membranes for each genotype and substrate). (F) Examples of images of myotube formation at day 2 of differentiation from WT and Mmp13^-/- myoblasts on BME and collagen. Sarcomeric myosin heavy chain (sMyHC) in red, phalloidin labeling actin in green, and DAPI labeling DNA. Scale bar = 50 μm. (G) The fusion index (percentage of nuclei in myotubes of all nuclei) is not changed between genotypes at any timepoint or substrate. There is a main effect of collagen substrates with less differentiation than BME substrates (N=3 wells per genotype per substrate per day). The * represents p<0.05 difference between WT and Mmp13^-/-;† represents a significant difference within each genotype of serum conditions or substrate.