DJ-1 in Mitochondrial Dynamics Figure 7. Mitochondrial and lysosomal colocalization. (A) To analyze mitochondrial and lysosomal colocalization DJ-1 KO and DJ-1 WT MEF were treated with EBSS medium and stained with 200 nM MitoTracker green and 200 nM LysoTracker red for 15 min at 37uC. Pictures were taken by life cell imaging microscope (Cell Observer Z1, Zeiss, Germany) at 37uC using ApoTomeH optical slides with 0.35–0.40 z-stacks and colocalization was determined with AxioVision4.7 software (Zeiss, Germany). (B) Statistical evaluation of the scatter-plot analysis of MitoTracker green and LsoTracker red co-staining (AxioVision4.7 software, Zeiss, Germany). The experiment was performed in duplicate analysing a total of 40 cells for each cell line (*p,0.001, Student’s t-test). (C) Fibroblasts from carriers of the E64D family and a healthy control were analyzed by live cell imaging using the same protocol for mitochondrial and lysosomal staining as described for MEF cells. Statistical evaluation mitochondria colocalizing with lysosomes compared to total mitochondria. We observed significantly reduced proportion of mitochondria colocalizing with lysosomes in the homozygous mutation carrier compared to healthy control (*p,0.01, Student’s t-test). No significant difference was observed between heterozygous carriers and control. The experiment was performed in duplicate analyzing a total more than 30 individual cells for each proband. doi:10.1371/journal.pone.0009367.g007 PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9367