Enriched collagen XXV in the culture medium promotes the formation of myotubes with increased number of nuclei. (A) HEK293T cells were transiently transfected with a vector encoding collagen XXV fused to GFP (Col25a1-GFP) or GFP alone (GFP). Ion exchange chromatography of conditioned media (C.M.) was used to enrich Collagen XXV-GFP released in the former condition as assessed by Western blotting. The corresponding fractions obtained after chromatography of the GFP C.M. were pooled to be used as negative control in functional assays. Next, C2C12 cells were treated with 10 µL/mL of the fractions described above or with 0.5 ng/mL recombinant GFP (rGFP). (B–D) Representative immunofluorescence images of C2C12 cells treated four days in a low serum medium as described in A. Differentiating myoblasts and myotubes were marked by the MF20 antibody (green). Total nuclei were stained with Hoechst. Scale bar: 100 µm. (E) Distribution of myotubes with the corresponding number of nuclei in each culture condition described in A. *P < 0.05.