Fig. 3

Human FUS aggregates are insoluble, display pre-amyloid properties and recapitulate features of human FUS pathology. A Schematic overview of the timepoints at which FUS aggregation was analyzed using immunofluorescence-based assays and biochemical insolubility assays at 3 hours, 1 months and 6 months post-injection. B, C Representative confocal images of mFUS^KO/hFUS^R521H mouse brains injected either with HA-FUS^R495X fibrils at 3 hours, 1 months and 6 months post-injection (B) or with PBS at 6 months post-injection (C) immunolabelled using antibodies against the pre-amyloid oligomer marker A11 (red), HA after 3 hours post-injection (green) and FUS after 1 month and 6 months post-injection (green). Yellow arrows indicate co-localization between A11 and FUS cytoplasmic inclusions detected in fibril-injected mice. DAPI (blue) as nuclear counterstaining. Scale bars: 10 µm, inset: 5 µm. D, E Representative confocal micrographs of mFUS^KO/hFUS^R521H mouse brains injected with HA-FUS^R495X fibrils using ubiquitin/p62/TDP-43 (red) and FUS (green) antibodies after 1 month (D) and 6 months (E) post-injection. Yellow arrows indicate co-localization between either ubiquitin/p62/TDP-43 and FUS cytoplasmic aggregates. Scale bar: 10 µm, inset: 5 µm. F Experimental outline of the serial fractionation of brain homogenates derived from mFUS^KO/hFUS^R521H mice either PBS- or HA-FUS^R495X fibril-injected and R6/2 Huntington’s model mice as a positive control for FUS insolubility [58]. G Immunoblotting of the sequential biochemical fractions from mouse brains using anti-FUS, anti-TAF15 and anti-TDP-43 antibodies. Anti-GAPDH was used as loading control