FIGURE 5

SORBS1 correct splicing is mandatory to generate large AChR clusters. (a) Schematic representation of the exon‐skipping strategy using antisense oligonucleotide (ASO). hiPSCs‐derived myotubes from controls are terminally differentiated and transfected with 50 nM of ASO at 5 days. At Day 7 of differentiation, hiPSC‐derived myotubes are treated with 0.5 mg/mL of agrin for 6 h. (b) RT‐PCR analysis and quantification of SORBS1 exon 25 inclusion on total RNA extracts isolated from Ctrl, DM1 and ASO‐transfected cell lysates. (c) Representative immunofluorescence of hiPSC‐derived myotubes at 7 days of differentiation and after 6 h of agrin stimulation (0.5 mg/mL). Images are confocal Z projections. (d) Violin plots representing the distribution of AChR clusters area in μm²; Ctrl (n = 21) from three independent experiments; DM1 (n = 21) from three independent experiments; ASO (n = 21) from three independent experiments. p < 0.01 and p < 0.0001, one‐way‐ANOVA followed by Tukey's post hoc test. Bar plots representing the distribution of AChR cluster sizes divided into groups of 0–50 mm², 50–100 mm² and superior to 100 mm². N = 21 from three independent experiments. p < 0.001 and p < 0.0001, two‐way‐ANOVA. (e) Representative immunofluorescence of AChR clusters in Ctrl and ASO‐transfected hiPSC‐derived myotubes in high‐resolution confocal microscopy. (f) Fluorescence intensity profiles of AChR and SORBS1 in newly formed clusters following 6 h of agrin stimulation.