Fig. 1: BAG5 regulates Parkin recruitment to mitochondria.

a Representative confocal micrographs of U2OS GFP-Parkin cells transfected with dsRed or FlagBAG5 and treated with 20 μm CCCP for 60 min. Scale bar is 20 μm. b Quantification of the percentage of transfected cells displaying GFP-Parkin recruitment onto the mitochondria by assessing colocalization of GFP-Parkin with TOM20. A minimum of 150 cells per condition were counted in three independent experiments, and statistical significance was determined by student’s t test (*p < 0.05). c Western blot confirming reduction of endogenous BAG5 protein levels in transfected U2OS GFP-Parkin cells. d Representative confocal micrographs of 2 h live cell time-lapse imaging of U2OS GFP-Parkin cells treated with 20 μm CCCP and transfected with non-targeting control or BAG5 siRNA 48 h prior to imaging (see Video 1). e Quantification of GFP-Parkin recruitment to mitochondria by evaluating the percentage of cells displaying colocalization between GFP-Parkin and mito-RFP. At least 200 cells were scored per condition by an individual blinded to the treatment. The vertical bars represent the mean ± SEM for three independent experiments. Two-way ANOVA followed by Bonferroni post hoc test revealed siBAG5 transfected cells had a significantly greater percentage of cells displaying GFP-Parkin recruitment to the mitochondria at timepoints between 30 and 60 min inclusive (*p < 0.05, **p < 0.01,***p < 0.001). Scale bar is 50 μm. f. Representative confocal micrographs of U2OS GFP-Parkin cells transfected with human BAG5 siRNA and rat FlagBAG5 treated with CCCP for 60 min. Scale bar is 20 μm g. Quantification of the percentage of transfected cells displaying GFP-Parkin recruitment onto the mitochondria by assessing colocalization of GFP-Parkin with TOM20. A minimum of 50 Flag negative and Flag positive cells per condition were counted in three independent experiments, and statistical significance was determined by student’s t test (*p < 0.05). h Representative Western blot demonstrating knockdown of human FlagBAG5 but not rat FlagBAG5 by human BAG5 siRNA.