Figure 3.

Clustering of AChRs in C2C12 myotubes cultured on different human laminins. (a) Representative images of AChR clusters in myotubes that were cultured on Permanox slides coated with the indicated laminins. Scale bar = 150 μm. (b) Quantification of AChR clusters formed by myotubes that were grown on Permanox slides coated with the indicated laminins. Quantifications represent the average number of clusters. For all laminins except laminin-111, laminin-411, and laminin-421, the total number of clusters that were used for the analysis was> 300. For laminin-111, 216 clusters were used. For laminin-411, 25 clusters were used. For laminin-421, 30 clusters were used. The clusters were collected from five independent experiments performed on different days. (c) Different laminins induced similar levels of AChR-α1 subunit expression in myotubes. HEK293 cell lysates were used as a negative control. Tubulin was used as a loading control. (d) Schematic representation of laminin-111, 211, 411, 511. LN domains are shown in green, L4 domains in red, LF domain in yellow and LG domains in orange. (e) Representative images of AChR clusters in myotubes that were cultured on the indicated laminin combinations. (f) Quantification of AChR clusters formed by myotubes grown on indicated combinations of laminin isoforms. The total number of clusters used in the analysis was>160 for 121 + 221 and 211 + 221; for 121 + 211 it was 71 and for 111 + 221 it was 99. Quantifications represent the average number of clusters. Scale bar = 150 μm. *p < 0.05, **p < 0.005, ***p < 0.0005. For statistical analysis we used one way ANOVA with Dunnett test; laminin-111 used as a reference. Error bars represent SEM values. AChRs were visualized with α-bungarotoxin.