Sarcomere length measurements of muscle fibers cultured in 2D and 3D culture systems at days 1 and 7 of culture. (A) Quantification of resting sarcomere length of muscle fibers in 2D and 3D tracked over 7 days, which was used for normalization of muscle fiber width measurements in Fig. 1 D and Fig. 2 F. (B) Sarcomere length at peak contraction of muscle fibers cultured in 2D and 3D at days 1 and 7. Data are means ± SEM; large dots represent mean values per mouse, and small dots represent single muscle fibers. Statistics were performed using a repeated measure two-way ANOVA in panel A and two-way ANOVA in panel B, with P < 0.05 considered as significant with * = P < 0.05, ** = P < 0.01, and **** = P < 0.001.