Proteasome impairment enhances autophagy in Drosophila larvae. A) Depletion of Rpn2 increases punctate Lysotracker Red (LTR) staining in Lamp1-GFP marked larval fat body cell clones of starved animals relative to surrounding control cells. B) Rpn2 knockdown induces autophagy in fat body cell clones of well fed larvae, as LTR dots are only observed in GFP-positive RNAi cells but not in control cells. C) Silencing of genes encoding different proteasome subunits results in a statistically significant induction of punctate LTR in well fed conditions (u test, n = 5-7 per genotype, ** P < 0.01), and error bars denote standard error. D) Immunostaining reveals cell-autonomous activation of punctate endogenous Atg8a labeling in Rpn2 RNAi cells, representing autophagosomes. E) Statistical evaluation of the effect of proteasome subunit RNAi on punctate endogenous Atg8a. Statistically significant differences are marked (u or t test, n = 5 per genotype, ** P < 0.01), and error bars denote standard error. (F) Western blots show that RNAi knockdown of genes encoding different proteasome subunits greatly increases the levels of both the specific autophagy cargo p62 and autophagosome-associated lipidated Atg8a-II. Numbers refer to relative expression levels compared to Tubulin, as determined by densitometric evaluation. Boxed areas in A, B and D are shown enlarged. Scale bar in A equals 20 μm for A, B, D.