DJ-1 in Mitochondrial Dynamics Figure 5. Assessing basal autophagy by LC3 protein monitoring in DJ-1 WT and DJ-1 deficient MEF. (A–C) LC3 protein monitoring reveals reduced basal autophagy in DJ-1 KO MEF. (A) WT (MEF DJ-1 +/+) or DJ-1 KO MEF (MEF DJ-1 2/2) were either left untreated (mock treatment, control) or were treated with medium lacking amino acids (EBSS) in the absence or presence of lysosomal inhibitors (PI) in the culture medium. Total protein extracts were analyzed by anti-LC3 WB. The increase of LC3-II normalized over GAPDH levels was quantified. (B, C) WT (MEF DJ-1 +/+) or DJ-1 KO MEF (MEF DJ-1 2/2) were pretreated with LY290042 for 3 hrs to reduce autophagy to a minimum basal level. Thereafter, in the presence or absence of lysosomal inhibitors (protease inh.) control medium was added to analyze basal autophagy, or EBSS was added to induce autophagy for the indicated period of time (min.). LC3 and GAPDH protein levels from total protein extracts are shown. (B) The decrease in basal autophagy is presented in the lower panel graph as follows. From three independet sets of experiments shown in C (boxed lanes in the upper and lower panels) and B (boxed lanes in the upper panel) were used to quantify the LC3-II signal intensity normalized over GAPDH. The measurements for DJ1 +/+ MEFs were set to one. The LC3-II signal intensity in DJ1 2/2 MEFs was compared to DJ1 +/+ MEFs (p-value 0.000676). (D) Although basal autophagy was reduced in DJ1 2/2 MEFs, autophagy was not abolished as further shown by transient expression of GFP-LC3 and detecting GFP-LC3 puncta as an indicator for autophagosomes. doi:10.1371/journal.pone.0009367.g005 maintained in MEF cells devoid of DJ-1 [36]. Next, we treated cells with the PI-3 kinase inhibitor compound LY294002 for 3 hours. Thereby, PI(3)P generation by Vps34/Beclin1 is inhibited and basal autophagy is reduced to a minimum. Under such circumstances, we compared basal autophagy in a time course after the LY294002 compound was removed and substituted with growth medium (Basal) or amino acid-free medium (EBSS) with or without protease inhibitors (Fig. 5B, 5C). In all cases from the three independent experiments presented in Fig. 5B (upper panel) and Fig. 5C (upper panel, lower panel), LC3-II protein abundance was reduced in the DJ-1 KO cells, but autophagic activity was not completely abolished (compare LC3-II signal intensities with and without the addition of protease inhibitors) as observed before. Normalized basal autophagy (0 min. time point in time courses from three independent experiments, boxed lanes in Fig. 5B, C) was quantified by setting the LC3-II signal intensities in DJ-1 WT cells to 1. The decrease in LC3-II signal intensity in DJ-1 KO cells was found to be significant with a p-value of 0.000676 (Fig. 5B, lower panel). In order to confirm that autophagy is not completely blocked, although the basal level was reduced in mutant DJ-1 cells, we transiently transfected DJ-1 WT and KO MEFs with GFP- LC3 and found puncta formation in both cell types (Fig. 5D). In support, by measuring the induction of autophagy by human GFP-WIPI-1 puncta formation (here: transient GFP-WIPI-1 puncta formation) we demonstrate that autophagy is not principially inhibited by the loss of DJ-1 (Fig. 6A and B). No difference in the amount of GFP-WIPI-1 puncta positive cells as a measure for autophagic activity was observed in either of the PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9367