Figure 4: Collagen VI has a direct effect on SC in vivo.

(a) Schematic diagram of the experimental approach for grafting SCs in Col6a1^–/– regenerating muscles. One day after cardiotoxin injury, 3,000 freshly isolated wild-type SCs or Col6a1^–/– SCs expressing the GFP-LC3 fusion protein were injected in regenerating Col6a1^–/– TA. Muscles were analyzed after 7 days from cardiotoxin injury. (b) Real-time PCR analysis of GFP expression in Col6a1^–/– muscles derived from 7-day cardiotoxin-injured mice without grafting (−) or grafted with wild-type or Col6a1^–/– SCs expressing GFP-LC3. Data are shown as mean±s.e.m. of three independent replicates (*P<0.01; unequal variance Student’s t-test; n=5 animals, each group). (c) Real-time PCR analysis of Col6a1 expression in Col6a1^–/– muscles derived from 7-day cardiotoxin-injured mice without grafting (−) or grafted with wild-type SCs or Col6a1^–/– SCs. Data are shown as mean±s.e.m. of three independent replicates (**P<0.01; n.s., not significant; unequal variance Student’s t-test; n=5 animals, each group). (d) Quantification of SCs, calculated as the number of SCs on 100 fibers, in Col6a1^–/– muscles derived from 7-day cardiotoxin-injured mice without grafting (−) or grafted with 3,000 wild-type SCs or Col6a1^–/– SCs. Data are shown as mean±s.e.m. of three independent replicates (**P<0.01; * P<0.05; n.s., not significant; unequal variance Student’s t-test; n=5–6 animals, each group). (e) Left panel, representative images of haematoxylin-eosin staining of TA cross-sections from Col6a1 ^–/– mice 7 days after cardiotoxin injury without grafting or grafted with wild-type SCs or Col6a1^–/– SCs. The asterisks label some areas still in the degeneration phase. Scale bar, 50 μm. Right panel, quantification of myofiber mean cross-sectional area (CSA) in Col6a1^–/– TA 7 days after cardiotoxin injury without grafting (−) or grafted with wild-type SCs or Col6a1^–/– SCs. Data are shown as mean±s.e.m. of three independent replicates (**P<0.01; unequal variance Student’s t-test; n=5–6 animals, each group). Ctx, cardiotoxin; KOSC, Col6a1^–/– SCs expressing GFP-LC3 protein; WTSC, wild-type SCs expressing GFP-LC3.