Fig. 7: iPSC-MNs expressing expanded G4C2 repeats are characterized by a decreased number of PML-NBs compared to gene corrected iPSC-MNs.

A C9-ALS and GC iPSCs were differentiated into motor neurons. Cells were fixed and stained with an antibody specific for neuron-specific class III beta-tubulin (TuJ1) and Choline Acetyltransferase (ChAT). Nucleic acid was labeled using DAPI. B, C C9 and GC iPSCs were differentiated into motor neurons. C9-ALS and GC iPSC-MNs were either left untreated or treated with sodium arsenite (500 μM) for 1 hr; where indicated cells were let to recover in drug-free medium for 2 hrs after treatment ( + rec.). Cells were fixed and stained with an antibody specific for PML. Nucleic acid was labeled using DAPI. PML-NBs were automatically segmented and quantified. The average number of PML-NBs is shown in (B). The average size of PML-NBs is shown in (C). N = 5 independent experiments, +/− s.e.m. Number of cells analyzed/condition: GC, control (8022); C9-ALS, control (6128); GC, arsenite (7188); C9-ALS, arsenite (8691); GC, arsenite + rec. (4997); C9-ALS arsenite + rec. (7829). One-way ANOVA, Bonferroni-Holm test. D, E GC and C9-ALS iPSC-MNs were either left untreated or treated with sodium arsenite (500 μM) for 1 hr; where indicated cells were let to recovered in drug-free medium for 2 hrs after treatment ( + rec.). Cells were fixed and stained with antibodies specific for the SG markers TIAR or G3BP1. Nucleic acid was labeled using DAPI. Scale bar is 20 μm. Representative images are shown in (D). SGs were quantified. The percentage of SGs/cell is shown in (E). N = 3–4 independent experiments, +/− s.e.m. Number of analysed cells/condition: GC, control (363); C9-ALS, control (340); GC, arsenite (1805); C9-ALS, arsenite (935); GC, arsenite + rec. (912); C9-ALS arsenite + rec. (885). One-way ANOVA, Bonferroni-Holm test.