α-synuclein accumulation in PARK2 iPSC-derived neurons. (A–C) Triple labeling for α-synuclein (red), tyrosine hydroxylase (TH; cyan), and βIII-tubulin (green) along with Hoechst (blue) staining of control A (B7), control B (WD39), Cent1-8, and PARK2 (PA9 and PB20) iPSC-derived neurons. (A) Arrows indicate α-synuclein+/TH+/βIII-tubulin+ neurons; arrowheads indicate α-synuclein+/TH-/βIII-tubulin+ neurons. Note the presence of α-synuclein+/TH-/βIII-tubulin- non-neural cells (asterisks). (B) High magnification confocal projection image of an α-synuclein (magenta)/βIII-tubulin (green) double-positive PA9 iPSC-derived neuron. (C) The proportion of α-synuclein+/βIII-tubulin+ neurons relative to βIII-tubulin-positive neurons was significantly higher in PA (PA1, 9 and 22) iPSC-derived neurons than in control A (B7 and YA9), control B (WD39) and Cent1-8 iPSC-derived neurons. Scale bars: A, 50 μm; C, 5 μm. ** indicates P < 0.01; * and ¶ indicate P < 0.05 (Mann–Whitney U-test). Data represent the mean and SEM of at least three experiments for each group.