Fig. 5. Recombinant human annexin V (AnxV) inhibits myotube formation in differentiating C2C12 and H9C2 muscle cell cultures. C2C12 cells were induced to differentiate in DM when the cultures were at 25% (not shown) and 50% confluency (A1,A2), and H9C2 cells at 95% confluency (B1,B2). In samples cultured in the presence of M1234 (not shown) or in the absence of annexin V (ctrl) myotube formation could clearly be observed in C2C12 cells at day 5 (DM d5, A3, arrows) and H9C2 cells at day 11 (DM d11, B3, arrows), and to a lesser extent in cells cultured in DM containing 100 µg/ml of recombinant human annexin V (A4,B4, arrows). Note that cells cultured in GM differentiate spontaneously and form myotubes when reaching 100% confluency (e.g. B1,B2, arrows). To assess the effect of annexin V on myotube formation quantitatively, multinucleated cells and their nuclei were counted in C2C12 cultures at DM d5 (A5) and H9C2 cultures at DM d11 (B5), after staining of DNA with DAPI (blue), and with titin (green) to detect differentiating cells. The quantitative results are shown in Fig. 6 and Table 1. Bar, 100 µm (A1-4,B1-4); 25 µm (A5,B5).