The mCherry-GFP-LC3 assay and elevated phagophore formation showing enhanced induction of autophagy by BRUCE depletion.

Mouse embryonic fibroblasts (MEFs) expressing mCherry-GFP-LC3 were transfected with control siRNA (siCtrl) (A and B) or siBRUCE (C and D) followed by treatment with rapamycin (Rap) (B, 2 μM, 4 hr) or CQ (D, 200 μM, 2 hr). After cell nucleus staining with DAPI, cells were examined for autophagy flux utilizing confocal microscopy. Autophagosomes in the cytoplasm emit yellow fluorescence versus red when inside lysosomes. Rapamycin (removal of the inhibitory role of mTOR on autophagy) served as a positive control for enhanced autophagy function (i.e., increased both yellow and red fluorescence dots). (E and F) Endogenous ATG16L was immunostained using an anti-ATG16L antibody in U2OS-shBRUCE-GFP-LC3 cells with BRUCE expressed (-DOX) or depleted (+DOX). (G and H) Quantifications of autophagosomes (yellow dots) and autolysosomes (red dots) in Fig 2A–2D. *, p<0.05. (I) Quantification of ATG16L dots in Fig 2E and 2F. Bar: 20 μm. *, p<0.05.