Additional file 3 of Tetraspanin CD82 is necessary for muscle stem cell activation and supports dystrophic muscle function

Additional file 3: Supplementary Figure 3. Delayed fusion in CD82-/- myoblast cultures is not due to early apoptosis/or reduced cell adhesion. (A, B) Immunofluorescence staining of cultures for MyoD (red) and H2Aγ (green) 72 hrs post-extraction from WT (A) and CD82-/- (B) animals. Arrowhead in (A) points at a MyoD+H2Aγ+ cell. (C) Quantification of H2Aγ+ cells and (D) non-adherent cells revealed no significant differences between the genotypes at all timepoints analyzed. (E) Myogenic cells from WT and (F) CD82-/- mice were plated at the same density and induced to form myotubes. At day 3 and 5 following differentiation, WT and CD82-/- cultures were immunostained for myosin heavy chain (red staining in G, H, L, M) to quantify the fusion index. The purity of the cultures assessed by MyoD staining was >80-90%. At day 3 there was no difference in myotube formation between cultures, as assessed by fusion index following staining with MF20 (I). (N) Quantification of fusion index after 5 days in differentiation media showed significantly decreased fusion in CD82-/- compared to WT cultures. Fusion index was calculated as the ratio of number of nuclei fused in MHC-myotubes over the number of total nuclei (****p<0.0001).