Influence of muscle fiber adhesion on contractility. (A) Representative confocal maximum intensity projection images taken with a 60× objective stained for α-dystroglycan (green) and nuclei (blue) in muscle fibers cultured for 1 and 7 days in 2D and 3D. Since treatment with collagenase destroys most membrane proteins, fibers are outlined with a dashed line at day 1. (B) Representative confocal maximum intensity projection images taken with a 60× objective stained for dystrophin (magenta) and nuclei (blue) in muscle fibers cultured for 1 and 7 days in 2D and 3D. (C) Quantification of mean α-dystroglycan fluorescence intensity in muscle fibers cultured for 1 and 7 days in 2D and 3D. (D) Quantification of mean dystrophin fluorescence intensity in muscle fibers cultured for 1 and 7 days in 2D and 3D. (E–G) Contractile measurements of muscle fibers cultured for 7 days in 2D, in 2D followed by dissociation with trypsin, or in suspension. (E) Quantification of resting sarcomere length of muscle fibers at day 7. (F) Quantification of the percentage of sarcomere shortening of muscle fibers at day 7. (G) Quantification of the contractile velocity of muscle fibers at day 7. Data are means ± SEM; large dots represent mean values per mouse, and small dots represent single muscle fibers. Significance was determined using one-way ANOVA with P < 0.05 considered as significant with ** = P < 0.01.