Fig. 1

Exogenously applied SOD1 aggregates enter cells and induce endogenous SOD1 aggregation. a Left panel Quantitative analysis of SOD1 association with NSC-34 cells using flow cytometry. Cells were either incubated with PBS (grey) or aggregated wtSOD1 (60 min incubation; blue line). Right panel Western blot of cell lysates detecting human SOD1 (and actin as a loading control). (b) Left panel Association with cells was quantified using flow cytometry. NSC-34 cells were treated with aggregated human SOD1 protein for 30 min and subsequently detected using immunofluorescence. Results shown are means ± SE, n = 3, ** p < 0.01. Right panel Confocal laser scanning micrograph of aggregated  wtSOD1 interacting with NSC34 cells after 30 min on ice to slow endocytosis. White dotted line represents cell membrane. (c) Confocal laser scanning micrographs of biotinylated wtSOD1 aggregates incubated with NSC34 cells for 60 min then either permeabilized with Triton x-100 or not, and subsequent detection using SA-Alexa488. (d) NSC-34 cells were transfected with wtSOD1-GFP and then incubated with either PBS, wtSOD1 (non-aggregated) or aggregated SOD1 and the number of cells with inclusions counted at 72 h. Results shown are means ± SE, n = 3, * p < 0.05. A minimum of 150 cells were counted per treatment and the average % of transfected cells with inclusions calculated across a minimum of 5 fields of view per treatment. (e) Cell lysates were further analyzed by filter trap assay. Any trapped SOD1-GFP material was measured using an anti-GFP antibody to avoid measuring aggregated material added to cells. Quantification of filter trap assays using a densitometer. Values are the mean intensity of trapped aggregated material averaged over 3 experiments. Results shown are means ± SE, n = 3, ** p < 0.01. (f) Exogenously added and endogenously produced SOD1 aggregates do not substantively colocalise. G93A SOD1 aggregates were labelled with Alexa-633 and added to NSC-34 cells expressing wtSOD1-GFP. After 48 h the cells were imaged using laser scanning confocal microscopy. Little colocalisation of Alexa633 and GFP signal was observed