DJ-1 Inhibits Formation of αSyn Intracytoplasmic Inclusions

<div><p>(A–L) CAD murine neuroblastoma cells were transfected with WT DJ-1 (A–F), L166P DJ-1 (G–I) or vector control (J–L), along with Flag-αSyn (D–L) or vector control (A–C) and differentiated in vitro by serum withdrawal for 72 h. Cells were fixed and stained with a mouse monoclonal antibody for αSyn and ToPro3, a nuclear dye, and images were obtained by confocal microscopy. Transfection of Flag-αSyn induced formation of intracytoplasmic inclusions (arrows). Scale bar, 20 μm.</p> <p>(M) Quantification of cells with inclusions was performed on ten random images from each of three wells per condition. Images were quantified by an observer blinded to the experiment. A significantly lower percentage of cells harbor inclusions in the context of WT DJ-1 overexpression. Aggregation is expressed as the percentage of cells containing αSyn aggregates per frame. Total cell number per frame, as determined by ToPro3 staining, did not differ significantly (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#sg003" target="_blank">Figure S3</a>). Data are shown as the mean ± SEM, and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p> <p>(N–S) Cells were fixed and stained with a monoclonal antibody for αSyn and a polyclonal antibody that recognizes both transfected human DJ-1 and endogenous murine DJ-1. DJ-1 does not appear to colocalize with the αSyn aggregates. Scale bar, 20 μm.</p>