LC3 turnover assay showing an elevated autophagy activity induced by BRUCE depletion.

(A) U2OS-shBRUCE-GFP-LC3 cells (clone #16, maintained in complete medium) were left untreated or treated with DOX to induce knockdown of BRUCE expression, followed by immunostaining with a LC3B antibody (green) and counter stained with DAPI (blue), Bar: 20 μm. (B) Semi-quantification of LC3B puncta numbers (immunofluorescent puncta) in each cell. *, p<0.05. (C) U2OS-shBRUCE-GFP-LC3 cells were transfected with control (siCtrl) and two distinct siRNAs targeting BRUCE (siBRUCE#2 and siBRUCE#3). LC3B puncta were immunostained and the results were quantified in (D). *, p<0.05. Bar: 10 μm. (E) BRUCE knockdown was performed with lentivirus mediated shRNAs (shScramble and shBRUCE) in starved human liver cell line THLE2. LC3B puncta were immunostained and the results were quantified (F). *, p<0.05. Bar: 20 μm. (G) DOX treated U2OS-shBRUCE-GFP-LC3 cells were incubated with CQ (50 μM, 2 hr) to block lysosome fusion and the total cell lysates were immunoblotted with indicated antibodies.