Fig 2: DJ-1 translocates to depolarized mitochondria in close proximity with optineurin in wild-type fibroblasts and neurons. (A and B) Control (Ctrl1) fibroblasts were treated with DMSO or valinomycin (Val., 1 µM) for the indicated time and immunostained for endogenous DJ-1 and the mitochondrial marker ATP5F1B. Nuclei were stained with TOTO-3. Arrowheads indicate examples of DJ-1 puncta that colocalize with mitochondria. (B) Quantification of the percentage cells with DJ-1/ATP5F1B colocalization (n = 3). For comparison, the time course of GFP-optineurin (GFP-OPTN)/ATP5F1B colocalization is also shown (n = 3). *P < 0.005 compared with the 3, 6, 12 and 24 h Val. condition for DJ-1/ATP5F1B colocalization. $P < 0.0001 compared with the 3 and 6 h Val. condition for GFP-OPTN/ATP5F1B colocalization. (C and D) Ctrl1 neurons were treated with Val. (1 µM) for the indicated time and immunostained for endogenous DJ-1, ATP5F1B and tyrosine hydroxylase (TH). Arrowheads indicate examples of DJ-1 puncta that colocalize with mitochondria. (D) Quantification of the percentage of TH-positive cells with DJ-1/ATP5F1B colocalization (n = 3). *P < 0.05 compared with the 3 and 12 h Val. condition. (E and F) Ctrl1 neurons were treated with DMSO or Val. for 6 h, followed by subcellular fractionation and western blotting of total (Tot.), cytosolic (Cyt.) and mitochondrial (Mit.) fractions for endogenous DJ-1, ATP5F1B and the non-mitochondrial protein COPB1. Equal amounts of protein (15 µg) were loaded for all fractions. This amount of 15 µg corresponded to nearly the entire mitochondrial fraction and to only ~5% of the cytosolic fraction, explaining why the ~2-fold increase in mitochondrial DJ-1 after valinomycin treatment in Ctrl1 cells (E and F) did not result in a visible decrease of DJ-1 in the cytosolic fraction. (F) Quantification of DJ-1/ATP5F1B (normalized to DMSO) in the mitochondrial fraction (n = 6). *P < 0.0001 compared with DMSO. (G and H) Ctrl1 fibroblasts were transfected with GFP-OPTN and after 24 h cells were treated with DMSO or Val. (1 µM) for the indicated time and immunostained as indicated. Arrowheads indicate examples of colocalization. (H) Percentages were quantified of cells with colocalization of DJ-1/GFP-OPTN/ATP5F1B. *P < 0.0001 compared with the 3 and 6 h Val. condition. (I and J) Ctrl1 fibroblasts were treated with DMSO or Val. for 6 h, followed by proximity ligation (PLA) with antibodies against endogenous OPTN and DJ-1 and immunostaining for the mitochondrial marker HSP60. Arrowheads indicate examples of PLA dots that colocalize with mitochondria. (J) Quantification (n = 3). *P < 0.0001 compared with DMSO. Scale bars = 5 µm. (K) Ctrl1 fibroblasts were transfected (Transf.) with empty vector (EV) of FLAG-tagged DJ-1, as indicated, and treated with DMSO or Val. for 6 h. After coimmunoprecipitation with anti-FLAG beads, the input and immunoprecipitate (IP) were analysed by sodium dodecyl-sulphate–polyacrylamide gel electrophoresis and western blotting using the indicated antibodies. The shown example is representative of six experiments.