Fig 2: Loss of MuSK, phosphoMuSK and Rapsyn in SOD1G93A fibers in vivo.(A) Representative confocal images of fibers in SOD1WT and SOD1G93A isolated fibers stained for α-BTX (magenta), rapsyn (green), MuSK (red) and DAPI (blue). Myofiber boundaries are represented as dash lines. (B) Representative confocal images of fibers in SOD1WT and SOD1G93A isolated fibers stained for α-BTX (magenta), rapsyn (green), phosphoMuSK (red) and DAPI (blue). Myofiber boundaries are represented as dash lines. (C) Mean intensity of MuSK signal in SOD1WT vs SOD1G93A isolated fiber. (D) Mean intensity of Rapsyn signal in SOD1WT vs SOD1G93A isolated fiber. (E) Mean intensity of phosphoMuSK signal in SOD1WT vs SOD1G93A isolated fiber. Scale bar 10 μm.

Fig 3: SOD1G93A in-vitro differentiated myofibers show similar defects to shDHC myofibers.(A) Quantification of peripheral nuclei in WT and SOD1G93A myofibersat days 6 and 9 of differentiation, in control and shDHC at day 9 of differentiation. (B) Quantification of myofibers with triads in WT and SOD1G93A conditions at days 6 and 9 of differentiation, and in control and shDHC at day 9 of differentiation. (C) Quantification of myofiber thickness in WT and SOD1G93A conditions at days 6 and 9 of differentiation, and in control and shDHC transfected myofibers at day 9 of differentiation. (D) Quantification of number of AChR cluster per fiber and AChR cluster length in WT and SOD1G93A conditions at days 6 and 9 of differentiation. (E) Representative epifluorescence images of WT myofibers at day 9 stained for AChR with BTX (green), MuSK (Red) and DAPI (blue). (F) Representative images of SOD1G93A myofibers at day 9 stained for α-BTX (green), MuSK (Red) and DAPI (blue). (G) Representative epifluorescence images of WT myofibers at day 9 stained for α-BTX (green), phosphoMuSK (Red) and DAPI (blue). (H) Representative epifluorescence images of SOD1G93A myofibers at day 9 stained for α-BTX (green), phosphoMuSK (Red) and DAPI (blue). Scale bar 10 μm.

Fig 4: Dynein is involved in MuSK localization at cell periphery and clustering at NMJ and microtubules are important for stabilization of NMJ.(A) Representative confocal images of day 9 myofibers transfected with control GFP plasmid (Control, top) or DHC shRNA (shDHC, bottom) and stained for MuSK. Arrows show MuSK at cell periphery. Note that MuSK localization at the cell periphery is reduced in shDHC-transfected myofiber. (B–D) Representative epifluorescence images of myofibers: not treated with ciliobrevinD ((B); control), treated with ciliobrevinD from Day 1 to Day 6 of differentiation (C, ciliobrevinD) or after ciliobrevinD washout at Day 4 of differentiation ((D), washout) as indicated. All conditions were fixed and stained at Day 6 of differentiation. Myofibers are stained for AChR with BTX (green), MuSK (red) and DAPI (blue). Arrowheads show MuSK localization at cell periphery. (E) Representative epifluorescence images of myofibers in control conditions stained for AChR with BTX (green), MuSK (red) and DAPI (blue).(F) Representative epifluorescence images of myofibers treated with nocodazole at Day 4 of differentiation for 2 hours (1 μg/ml) stained for α-BTX (green), MuSK (Red) and DAPI (blue). Arrows show MuSK localization at cell periphery. (G) Representative epifluorescence images of myofibers treated with nocodazole from Day 4 to 7 of differentiation (0.25 μg/ml) stained for α-BTX (green), MuSK (Red) and DAPI (blue). Scale bar (A) 20 μm. Scale bar (B–G) 10 μm.