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. 2017 Jul 4;6:e18459. doi: 10.7554/eLife.18459

Figure 4. daf-2 insulin/IGF-1 receptor mutants display increased autophagic activity in most tissues.

(A–D) Quantification of autophagosomes (AP) and autolysosomes (AL) in adult Day 1, 3, 5, 7, and 10 daf-2(e1370) animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control, dark green lines) or Bafilomycin A (BafA, light green lines). Tissues examined were the intestine (A), body-wall muscle (B), pharynx (C), and nerve-ring neurons (D). The black dashed lines in (A–C) show data from wild-type (WT) control animals from Figure 3 for comparison (animals were analyzed in parallel). The black dashed line in (D) shows data from WT animals incubated at 20°C for their entire lifespan. Data are the mean ± SEM of ≥25 animals combined from three independent experiments. ∧, WT control vs. daf-2 control at Days 1, 3, 5, 7, and 10; *, daf-2 control vs. daf-2 + BafA at Days 1, 3, 5, 7, and 10; #, daf-2 control at Days 3, 5, 7, and 10 vs. daf-2 control at Day 1. ***/∧∧∧/###p<0.0001, **/∧∧/##p<0.001, */∧/#p<0.01 by Poisson regression. See also Figure 3—figure supplement 1A–H for quantification of APs in gfp::lgg-1 transgenic animals.

DOI: http://dx.doi.org/10.7554/eLife.18459.013

Figure 4.

Figure 4—figure supplement 1. Hypodermal seam cells in daf-2 mutants display increased autophagy, whereas lipidation-independent punctate structures are present in these cells in glp-1 mutants.

Figure 4—figure supplement 1.

(A–C) Quantification of autophagosomes (AP) (A–B) and autolysosomes (AL) (C) in hypodermal seam cells of adult Day 1 wild-type (WT), daf-2(e1370), and glp-1(e2141) transgenic animals expressing gfp::lgg-1 (A) or mCherry::gfp::lgg-1 (B–C) and injected with DMSO (control) or Bafilomycin A (BafA). Data are the mean ± SEM of ≥25 animals combined from at least three independent experiments. ***p<0.0001, *p<0.051 by one-way ANOVA. (D–E) Quantification of GFP-positive punctae in hypodermal seam cells of adult Day 1 WT and daf-2(e1370) animals raised at 20°C (D), and WT and glp-1(e2141) animals raised at 25°C (E) expressing WT gfp::lgg-1 (LGG-1) or mutant gfp::lgg-1 (LGG-1(G116A)). Data are the mean ± SEM from three independent experiments, each with ≥10 animals (one representative experiment shown). ****p<0.0001 by one-way ANOVA. (F–G) Quantification of APs and ALs in the hypodermal seam cells of Day 1 WT and daf-2(e1370 mutants expressing mCherry::gfp::lgg-1 raised at 20°C (F) or Day 1 WT and glp-1(e2141) mutants expressing mCherry::gfp::lgg-1 raised at 25°C (G) and fed from hatching with bacteria expressing empty vector (control) or dsRNA encoding rab-7. Data are the mean ± SEM of ≥40 animals combined from three experiments. ****p<0.0001 and **p<0.005 by one-way ANOVA. (H–I) Quantification of GFP-positive punctae in hypodermal seam cells of adult Day 1 WT, atg-3(bp412) (H) and atg-18(gk378) (I) animals expressing WT gfp::lgg-1 (LGG-1) or mutant gfp::lgg-1 (LGG-1(G116A)). Animals were raised at 20°C. Data are the mean ± SEM combined from three independent experiments for atg-3 with ≥22 animals combined from two independent experiments for atg-18 with ≥16 animals. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05 by one-way ANOVA.
DOI: http://dx.doi.org/10.7554/eLife.18459.014