Fig. 5

Organization of AChR clusters during maturation and Ca²⁺ response to acetylcholine. a Representative images of 2-, 4-, and 10-day-old myotubes cultured on nanopattern surface and immunolabeled for α-actinin (red) and α-bungarotoxin (α-BTX, green) together with a DAPI staining (blue). Lower panels are enlargement of the white rectangles. Scale bar: 20 μm. b, c, d Quantification of the average number of acetylcholine receptors (AChR) clusters per field (b), AChR cluster size (c), and of the average numbers of AChR clusters per myotube (d). Data were obtained from three independent experiments. Error bars are mean ± SEM. e−f Myotubes were loaded with 5 μM Cal520-AM to measure changes in cytosolic Ca²⁺, and stimulated with 10 μM of acetylcholine (ACh). Traces represent the mean ± SEM of Ca²⁺ responses of myotubes differentiated for 4 (e) and 10 days (e). g Statistical evaluation of the amplitude of the peak following ACh stimulation. Error bars are mean ± SEM