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. 2017 Aug 1;31(15):1561–1572. doi: 10.1101/gad.301648.117

Figure 4.

Figure 4.

Autophagy is dysfunctional in aged worms, and inhibition of its initiation increases life span. (A) Day 9 RNAi against genes involved in autophagosome regulation (I), induction (II), nucleation (III), expansion/maturation (IV), lysosomal fusion (V), and degradation (VI). Relative percentage change in MTL ± SEM compared with control is shown. Statistics are in Supplemental Table 3. Gene knockdown validation is shown in Supplemental Figure S4E. (B) Quantification of GFP::LGG-1 foci over time in hypodermal cells of rrf-3(pk1426);lgg-1p::GFP::lgg-1. Foci were quantified in young and old worms from images at 100× magnification. n = 50. Red lines show median and interquartile range. (****) P < 0.0001. (C) Representative Western blot of free GFP across life balanced to total GFP::LGG-1. The cleaved GFP bands correspond to the GFP::LGG-1 degradation products in the autolysosome. (D) Day 15 Quantification of GFP::LGG-1-positive foci in hypodermal cells of rrf-3(pk1426);lgg-1p::GFP::lgg-1 worms following day 9 RNAi knockdown. Foci were quantified from images with 100× magnification. n = 50. Red lines show median and interquartile range. (****) P < 0.0001. LGG-1 knockdown validation is shown in Supplemental Figure S4G. (E) Representative Western blot of free GFP (balanced to GFP::LGG-1) in young and old worms treated with chloroquine. The cleaved GFP bands correspond to the GFP::LGG-1 degradation products in the autolysosome. (F) Representative Western blot of day 9 RNAi against bec-1, vha-15, and lgg-1 and its effect on free GFP compared with GFP:LGG-1 measured at day 15. The cleaved GFP bands correspond to the GFP::LGG-1 degradation products in the autolysosome. (G) Day 9 inactivation of lysosomal degradation or acidification in rrf-3(pk1426) has no effect on MTL or the MTL increase mediated by bec-1 inhibition when used in combination. qPCR validation is shown in Supplemental Table S12. All Western blots used antibodies against GFP. Days represent days after first egg lay. Life span statistics are in Supplemental Tables S8 and S9.