Collagen XXV containing conditioned media promote fusion of myoblasts into myotubes. (A) C2C12 cells were transfected 48 h in a differentiation medium, with a vector encoding collagen XXV fused to GFP (Col25a1-GFP) then live cells were stained on ice with an anti-GFP antibody. After GFP staining, cells were fixed, permeabilized and stained with Hoechst (nuclei in blue). a’ and b’ correspond to cells in a and b respectively, with phase contrast microscopy. Scale bar: 15 µm. (B) Strategy used to culture C2C12 cells in differentiating medium in the presence (Collagen XXV-GFP) or absence (GFP) of the soluble ectodomain of collagen XXV. (C) Immunoblot analyses of 10T1/2 cells transiently transfected 48 h with a vector encoding GFP (GFP) or Col25a1-GFP. Left panel: cell lysate; right panel: culture media. Note that Collagen XXV-GFP fusion proteins (arrowhead) are abundant in the appropriate culture media and barely detectable in the cell lysate. The same gel presenting the different cell lysates is presented twice, one corresponding to a low exposure (left), the other one a high exposure (right). 20 µg proteins from cellular extracts and 20 µg proteins from each culture medium were loaded. * non specific band. (D) Representative immunofluorescence images of C2C12 cells cultured four days in a low serum medium of transfected 10T1/2 cells, as described in B. Differentiating myoblasts and myotubes were marked by the MF20 antibody (green). Total nuclei were stained with Hoechst. Scale bar: 100 µm. (E) Fusion index of myoblasts in D, calculated as percentage nuclei in MyHC positive myotubes (≥3 nuclei) of total nuclei. (F) Distribution of myotubes with the corresponding number of nuclei in each culture condition described in B. n = 3 different experiments. *P < 0.05 vs GFP. (G) Expression of myod, myogenin and myf5 was analyzed by RT-qPCR in cells cultured 1 (DM1) or 2 days (DM2) in differentiation 10T1/2 conditioned media, as described in B or in classic differentiation medium (Ctrl). Data are means ± s.e.m of three different experiments. *P < 0.05; ***P < 0.001.