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 −/−) 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 −/−) 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 −/− MEFs was compared to DJ1 +/+ MEFs (p-value 0.000676). (D) Although basal autophagy was reduced in DJ1 −/− MEFs, autophagy was not abolished as further shown by transient expression of GFP-LC3 and detecting GFP-LC3 puncta as an indicator for autophagosomes.