Figure 5

Proteasome inactivation-induced autophagy requires Atg genes and sima/HIF-1α . A) Depletion of Rpn2 increases punctate LTR staining in larval fat body cell clones in fed animals, marked by membrane-associated mCD8-GFP. B) Simultaneous silencing of Rpn2 and sima results in a block of punctate LTR staining. C) Overexpression of dominant-negative (DN) Atg4 inhibits LTR dot formation in Rpn2 RNAi cells. D, E) Simultaneous silencing of Rpn2 and p62 using two independent RNAi lines combined (D) or a third independent single p62 RNAi line (E) does not inhibit punctate LTR staining in larval fat body cell clones in fed animals. F) Depletion of p62 blocks aggregate formation in Rpn2 RNAi cells. G) Statistical evaluation of punctate staining in Rpn2, Rpt1 and Prosβ2 knockdown cells. Depletion of sima using 3 independent RNAi transgenes, silencing of BNIP3, Atg1, Atg9, Atg12, or overexpression of dominant-negative Atg1, Vps34, Atg4 inhibits proteasome inhibition-induced autophagy. Depletion of p62 or Atg18b (which is dispensable for starvation-induced autophagy and used as a negative control here) does not block the autophagy-inducing effect of proteasome inactivation. Statistically significant differences are marked (Kruskal-Wallis test or ANOVA, n = 5-8 per genotype, * P < 0.05, ** P < 0.01), and error bars denote standard error. H) Similarly, silencing of sima but not p62 attenuates LTR puncta formation in Prosα1 and Prosα5 RNAi cells. Statistically significant differences are marked (Kruskal-Wallis test or ANOVA, n = 5-8 per genotype, * P < 0.05, ** P < 0.01), and error bars denote standard error. Boxed areas in A-F are shown enlarged. Scale bar in A equals 20 μm for A-F.