Figure 2: Proliferation and fusion assays. (A) Cell proliferation assay at 48 and 96 hours. No differences in proliferation were observed between untransduced, mock transduced and MMP-9 transduced cells. (B) Cell fusion assay at 48 and 96 hours. No differences in the fusion index were observed between untransduced, mock transduced and MMP-9 transduced cells. Two independent experiments were tested with n = 3 for each condition

Figure 4: Invasion assay. 2.1×10⁵ cells in a serum-free proliferation medium were added to the upper compartment whereas the lower chamber was filled with proliferation medium. The bottom of the insert is an 8 µm pore size polycarbonate membrane covered by a layer of collagen matrix. Following a 72-hours incubation period, the inserts were removed, washed and colored. (A) The shown pictures are representative of the experiment and illustrate some of the analyzed pictures (where the backgrounds were removed). (B) Colorimetric analysis of cell density. For each picture, the area of points was measured using the Scion Image software giving the cell density. A significant difference was observed between untransduced and MMP-9 transduced MPCs with *p < 0.001. CTL: untransduced MPCs, MMP-9: MMP-9 transduced MPCs. Two independent experiments were tested with n = 4 for each group and for each experiment. 

Figure 6: Single perpendicular MPC transplantation per muscle. (A) Representation of the number of dog dystrophin-positive fibers in function of the position along the TA muscle. Each position corresponds to a cryostat section. Each section is separated by 120 µm from the previous one. (B) Evaluation of the spreading of transplanted MPCs. The number of cryostat sections which contained at least 1 dystrophin-positive fiber was counted. A significant difference was observed between the spreading of untransduced and of MMP-9 transduced MPCs with *p < 0.001. (C) Total number of dystrophin-positive fibers counted on all cryostat sections. A significant difference was observed between the transplantation of untransduced and of MMP-9 transduced MPCs with dp < 0.05. n = 4 for each group. 

Figure 1: MMP-9 over-expression. (A) Western blot with 20 µg of protein. Two bands corresponding to MMP-9 products were observed in MMP-9 transduced MPCs. (B) Zymography with 15 µg of protein. MMP-9 activity was increased in MMP-9 transduced MPCs compared to control cells. MMP-2 activity was similar in each cell group. U: Untransduced cells, M: Mock transduced cells, T: MMP-9 transduced cells (cells coming from 2 different transductions were used). 

Figure 3: Transwell migration assay. 3×10⁴ cells in a serum-free proliferation medium were added to the upper compartment while the lower compartment contained proliferation medium. The upper and lower compartments were separated by an 8 µm pore size polycarbonate membrane. After a 14-hours incubation period, the inserts were removed, washed and colored. (A) The pictures shown are representative of the experiment and illustrate some of the analyzed pictures (where the backgrounds were removed). (B) Colorimetric analysis of cell density. For each picture, the area of points was measured using the Scion Image software giving the cell density. No significant differences were observed between the cells over-expressing MMP-9 and the untransduced cells. CTL: untransduced MPCs, MMP-9: MMP-9 transduced MPCs. Two independent experiments were tested with n = 5 for each group and for each experiment. 

Figure 5: MPC transplantation. All the MPC transplantations were done in immunodeficient Rag^-/- mice. Ten perpendicularinjection trajectories were done in each muscle. (A,B,C) Cell transplantation in irradiated muscles. (D,E,F) Cell transplantation in muscles previously injected with cardiotoxin. (A,D) One month following cell transplantation, muscles were harvested and the graft success was evaluated by counting the number of dog dystrophin-positive fibers on each cryostat section. The number of dystrophin-positive fibers is the average of the 3 cross-sections having the highest number of dystrophin-positive fibers and separated by at least 240 µm. In both cases (in irradiated and cardiotoxin treated muscles), a significant difference was observed between the transplantation of untransduced and of MMP-9 transduced MPCs with *p < 0.01. (B,C,E,F) Fluorescent immunodetection of dog dystrophin (in red). (B,E) are representative cross-sections of the transplantation of untransduced MPCs and (C,F) are from muscles transplanted with MMP-9 transduced MPCs. Scale bar = 100 µm. The cross-sections represent the global result from the transplantation with n = 4 for each group