Fig 1: Nanoparticle-induced αS inclusions contained membrane-bound αS. Three groups of cells, WT/SiO2, GBA1-/SiO2, and GBA1-, described in Figure 6, were prepared for EM examination. Uranyl-lead EM staining revealed that only cells in GBA1-/SiO2 group contained abundant nanoparticle-associated inclusions (denoted by red arrows). The fields on the left panels were magnified in the middle panels and further in the right panels to reveal the presence of inclusions in the cells of GBA-/SiO2 group, and the inclusions contained a cluster of membranous structures in the absence of intact encircling membrane and filamentous structure (denoted by a red diamond), indicating that the endolysosomes in cells were ruptured and no αS fibril was formed. In contrast, cells in WT/SiO2 group contained abundant congregated nanoparticles which were well-confined in intact endolysosomes (denoted by a red cross), and those in GBA1- group contain abundant abnormally swollen membranous structures (denoted by a red star). Immuno-EM further showed that the nanoparticle-associated membranous structures in GBA1-/SiO2 group were immunolabeled by primary antibody against αS (NACP98, Mayo Clinic) (Dickson et al., 1999) and 18-nm gold conjugated secondary antibody (111-215-144, Jackson ImmunoResearch Laboratories) as denoted by a red arrowhead. Scale bar: 2 μm for the first column; 500 nm for the second column; 100 nm for the third and fourth column.