Fig. 2

Internalized aggregates entering via the endocytic pathway escape from the endosomes to the cytosol. a Confocal microscopy of the co-staining of wtSOD1 aggregates (20 μg/mL) taken up by NSC-34 cells after 10, 30 and 60 min at 37 °C, with Lysotracker Red. Fixed and permeabilized cells were immunostained with an anti-human specific SOD1 antibody. Bars represent 25 μm. Arrowheads indicate areas of localisation of SOD1 outside the acidic compartment. (b) Western blotting of cytosolic, membrane (ER/Golgi), nuclear and cytoskeleton fractions (10 μg) collected from NSC-34 cells treated with wtSOD1 aggregates (20 μg/mL) for 2 h at 37 °C. Western blots were stained for the presence of human SOD1, EEA1, actin and vimentin. (c) Digitonin selectively permeabilizes plasma membrane of NSC-34 cells. NSC-34 Cells were treated with either digitonin (10 μM) or Triton-x100 (0.5 %) and then immunostained for membrane bounded markers LAMP1, BIP and EEA and the cytosolic β-tubulin. Membrane bounded markers were not detected when permeabilized with digitonin indicating the specificity of digitonin for plasma membrane. Cells were counterstained with Red Dot 2. (d) Laser scanning confocal micrographs of NSC-34 cells were treated with aggregated human SOD1 protein for 60 or 120 min then fixed and permeabilized with either Triton-x100 (0.5 %) or digitonin (10 μM). SOD1 aggregates were detected using Alexa488 conjugated to streptavidin and counter stained with the nuclear dye Red Dot 2. SOD1 aggregates are only detected upon digitonin permeabilization after 120 min SOD1 incubation. White dotted line represents cell membrane