FM (Invitrogen, USA). Hoechst 33342 (Molecular Probes, USA) was used to stain nuclei. Analysis of mitochondrial morphology was done with an inverted Zeiss Axiovert microscope (Zeiss Plan- Apochromat 63x/1,4) with temperatured incubation chamber at 37uC. Time lapse pictures were taken with an AxioCamMRm camera (Zeiss, Germany) in 5 seconds intervals using AxioVision software (Zeiss, Germany). The series of pictures were saved uncompressed and analyzed with AxioVision software (Zeiss, Germany). For colocalization assays of mitochondria and lysosomes, additional to MitoTrackerH green, the cells were stained with 200 nM LysotrackerH red (Invitrogen, USA), according to the manufacturers instructions. To determine the degree of colocalization images were loaded into Axiovision 4.7.2 software and evaluated accordingly (Zeiss, Germany). Analysis of Mitochondrial Morphology Fluorescence microscopy images were optimized by adjusting the contrast and afterwards binarized by conversion to 8-bit images. After unspecific noise of the fluorescence signal was reduced, a spatial filter (convolution filter) as well as a threshold was applied to the images to define mitochondrial structures. Using Image J 1.41o software (Wayne Rasband; National Institutes of Health, USA), every single mitochondrion of the investigated cells was marked to analyze morphological charac- teristics such as its area, perimeter, major and minor axes. On the basis of these parameters, the aspect ratio (AR) of a mitochondrion (AR; ratio between the major and the minor axes of the ellipse equivalent to the object) and its form factor (FF; perimeter2/ 4p*area), consistent with the degree of branching, were calculated [21]. Electron Microscopy DJ-1 deficient and DJ-1 WT MEF were fixed in 2.5% glutaraldehyde in Hank’s modified salt solution (HMSS), postfixed in 1% OsO4 in 0.1 M cacodylate buffer, scraped off, centrifuged and dehydrated in a series of ethanol. The 70% ethanol step was saturated with uranyl acetate for contrast enhancement. Dehy- dration was completed in propylene oxide and the specimens were embedded in Araldite (Serva, Germany). Ultrathin sections were produced on a FCR Reichert Ultracut ultramicrotome (Leica, Germany), mounted on pioloform-coated copper grids and contrasted with lead citrate. Specimens were analyzed and documented with an EM 10A electron microscope (Zeiss, Germany). Western Blot Western Blot (WB) analysis were conducted as described previously [3]. Cells were lysed in buffer containing PBS, 1% Triton and protease cocktail (Roche, Germany). Lysates were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against Drp1 and OPA1 (BD Transduction Laborato- ries, USA), hFis (Axxora, Switzerland), Mfn2 and Actin (Sigma, Germany), phospho-p42/44 MAPK (Erk1/2) (Tyr202/Tyr204; CellSignaling Technology, USA), p42/44 MAPK (Erk1/2; CellSignaling Technology, USA), Prohibitin (NeoMarkers, Fre- mont, CA, USA), GAPDH (Thermo Scientific, Fremont, CA, USA). Secondary antibodies were purchased from GE Healthcare (UK). On each WB 50 mg of protein were loaded per lane. Assessing Autophagy: GFP-WIPI-1 Puncta Formation To conduct GFP-WIPI-1 puncta-formation analyses [22,23], DJ-1 WT or KO MEF were transiently transfected using Lipofectamin2000 (Invitrogen, USA) at a ratio of 1:2.5 (pEGFP. DJ-1 in Mitochondrial Dynamics C1-WIPI-1 DNA:Lipofectamin2000) for 48 hrs according to the manufactorer’s instruction. Routinely, transient transfections were controlled with empty vectors (data not shown). Autophagy was induced by incubating the cells 3 hrs (37uC, 5% CO2) with rapamycin (500 nM) or starvation medium (EBSS); or autophagy was inhibited by the administration of wortmannin (233 nM). For quantitative confocal microscopy of overexpressed GFP-WIPI-1, three independent prepared slides for each condition (100 cells/ slide) were analyzed. Confocal microscopy was conducted using Zeiss/Axiovert 100M/LSM510 and a 63x 1.4 DIC Plan- apochromat objective [24]. Assessing Autophagy: Endogenous LC3-II Protein Monitoring To conduct LC3-II protein monitoring [25] of endogenous LC3 in DJ-1 WT or KO MEF, cells were lysed with hot Laemmli buffer and subjected to anti-LC3 WB analyzes using LC3 Antibody (Nanotools, Germany). LY290042 pretreatment was performed using 10 mM LY290042 for 3 hrs. Protease inhibitors (Protease inhibitor complete, Roche) were added to the culture medium and renewed every 30 min. Results were normalized over GAPDH levels or the ratio of LC3-II/LC3-I was determined based on densitometry (Image Quant 5.1). Mitochondrial Fractionation Cellular fractionations were performed using a mitochondrial isolation kit for cultured cells (Pierce Rockford, USA), according to the manufacturer’s instructions. The fractions received, were analyzed by Western-Blot. The mitochondrial fraction was defined using an antibody against prohibitin, the cytosolic fraction was defined by using an antibody against GAPDH. Results Decreased Respiratory Rates in Intact Mouse Embryonal Fibroblasts from DJ-1 KO Mice To detect mitochondrial dysfunction by respirometry, we compared mitochondrial oxygen consumption in intact MEF cell lines from DJ-1 KO mice and littermate control cells. Measure- ments were performed with high resolution respirometry in freshly harvested intact cells with pyruvate as added substrate (Fig. 1A). Significantly diminished rates of endogenous respiration (233%) were observed in DJ-1 KO cells compared to the controls. Addition of oligomycin, an inhibitor of the F0F1ATPase, allowed to measure the nonphosphorylating respiration, which was the same in both types of cells. Then titration with the uncoupler FCCP revealed a typical activation followed by an inhibition of respiration. Rates of uncoupled respiration were significantly lower (up to 234%) in DJ-1 KO compared to WT cells (Fig. 1A). These results clearly demonstrate decreased endogenous oxidative phosphorylation and diminished uncoupled respiration of DJ-1 KO cells compared to WT controls in the presence of endogenous substrates plus the added complex I-dependent substrate pyruvate. To address whether or not specifically the complex I-dependent mitochondrial function is affected we investigated cell homoge- nates by means of a special substrate inhibitor titration protocol [23,26]. This protocol allows the sequential measurement of maximum phosphorylation rates (state 3) for two different substrates in the same sample. As shown in Fig. 1B addition of 5 mM ADP increased the respiration using pyruvate and malate as substrates (state 3pyr/mal). However the increase in state 3 respiration was significantly lower (250%) in DJ-1 KO cells compared to controls. After inhibition of the complex I-dependent respiration with rotenone the addition of complex II-dependent PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9367