FIGURE 2.

Confocal laser and electron microscopic analyses of α-syn inclusions in plasmid-derived α-syn-expressing cells treated with seed α-syn. A–D, confocal laser microscopic analyses of control SH-SY5Y cells transfected with pcDNA3 vector and Lipofectamine alone (A), cells transfected with pcDNA3-α-syn (WT) (B), cells transduced with the seed α-syn (Seed αS) (C), and cells transfected with both pcDNA3-α-syn and Seed αS (WT + Seed αS) (D), immunostained with anti-Ser(P)¹²⁹ (green), and counterstained with TO-PRO-3 (blue). The arrows indicate cytoplasmic round inclusions stained with anti-Ser(P)¹²⁹ (PSer129). Scale bars, 20 μm. E–F, comparison of confocal images of cells transfected with both α-syn plasmid and Seed αS (E) and tissue sections from DLB brains (F) using anti-Ser(P)¹²⁹ (green) and anti-ubiquitin antibodies (red). Cytoplasmic inclusions in transfected cells (arrows) are positive for ubiquitin, like Lewy bodies (arrowheads) in DLB brains. Scale bars, 20 μm. G and H, confocal microscopic images of cells transfected with both pcDNA3-α-syn and Seed αS. Cells were stained with 0.05% Thioflavin S (green) and anti-Ser(P)¹²⁹ antibody (red). The boxed area on the left is shown in the right panel. Scale bars, 20 μm on the left and 10 μm on the right. I and J, electron microscopic analyses of cells transfected with both pcDNA3-α-syn and Seed αS. High magnification of the boxed area in I is shown in J. An asterisk or arrow indicates a nucleus or mitochondrion, respectively. Scale bars, 2 μm in I and 200 nm in J. K, immunoelectron microscopic observation of cells transfected with both pcDNA3-α-syn and Seed αS using a polyclonal antibody against phosphorylated Ser¹²⁹ of α-syn. Scale bar, 200 nm.