Viral Transduction
Pseudotyped SARS-CoV-2 Lentivirus​
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To test whether SARS-CoV-2 entry depends on clathrin-mediated endocytosis, we used a pseudovirus system. Lentiviral particles pseudotyped with the SARS-CoV-2 spike protein were applied to HEK-293T cells stably expressing ACE2 following plasmid transfection. Infection efficiency was measured by GFP reporter expression. In parallel, cells were treated with siRNA targeting clathrin heavy chain (CHC) to disrupt clathrin-mediated endocytosis. Compared to control siRNA, CHC knockdown significantly reduced pseudovirus infectivity, demonstrating that clathrin-dependent pathways are essential for viral entry.
LAMP2 Knockdown in iPSC-derived Neurons​​
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To test the role of lysosomal proteins in Lewy body–like pathology, I knocked down LAMP2 in iPSC-derived dopaminergic neurons using shRNA. Western blotting confirmed efficient knockdown (A), and single-cell imaging revealed that neurons lacking LAMP2 developed visible α-synuclein inclusions, similar to those treated with IFN-γ (B). Confocal microscopy further showed that LAMP2-deficient neurons accumulated significantly more phospho-α-synuclein aggregates compared to scrambled controls (C).
Adenovirus expression of α-syn in neurons​​
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To determine whether α-synuclein expression and PFF internalization are interchangeable for inclusion formation, iPSC-derived dopaminergic neurons were generated in three genetic backgrounds: α-syn knockdown (Lenti), wild-type, and neurons engineered to overexpress α-syn-HA (Adenovirus). These cultures were then exposed to IFN-γ and monitored over time for the formation of phosphorylated α-synuclein inclusions. Only neurons treated with IFN-γ formed phosphorylated α-syn aggregates in the 14 day time period.
