Figure 2. Lysosomal acidity and degradation activity are increased in daf-2.

(A–H”) Confocal fluorescence images of the intestine in wild type (WT; A–D”) and daf-2(e1370) (E–H”) adults at different ages stained by LSG DND-189 and LTR DND-99. (I) The relative intensity of LSG/LTR in wild type and daf-2(e1370) at different ages was quantified. At least 10 animals were scored in each strain at each day. (J–L) Confocal fluorescence images of the hypodermis at adult day 2 in wild type (WT; J), daf-2(e1370) (K) and cup-5(bp510) (L) expressing NUC-1::pHTomato controlled by the heat-shock (hs) promoter. The average intensity of pHTomato per lysosome is shown in (M). At least 20 animals were scored in each strain. (N) Western blot analysis of CPL-1 processing in wild type (WT) and daf-2(e1370) at different adult ages. The percentage of mature CPL-1 was quantified (O). Three independent experiments were performed. In (I, M, O), data are shown as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (I, M) or two-way ANOVA with Fisher’s LSD test (O) was performed to compare all other datasets with wild type (M) or wild type at day 1 (I, O), or to compare datasets that are linked by lines. *p<0.05; **p<0.001. All other points had p>0.05. N.S., no significance. Scale bars: 5 μm.

Figure 2—figure supplement 1. pHTomato can be used to probe lysosomal acidity in C. elegans.

(A–B’’) Confocal fluorescence images of the hypodermis in wild type (WT) at day 1 (A–A’’) and day 5 (B–B’’) stained by LSG DND-189 and LTR DND-99. White arrowheads indicate vesicular lysosomes stained by both LSG and LTR; yellow arrowheads indicate an LTR-positive and LSG-negative vesicular lysosome. White arrows indicate lysosomal tubules stained by LTR but not LSG. (C, D) The relative intensity of LSG/LTR in the hypodermis (C) and the percentage of LTR-positive lysosomes stained by LSG (D) was quantified. At least 10 animals were quantified at each day. (E–G’’) Confocal fluorescence images of the hypodermis in wild type (WT, (E–E’’), daf-2(e1370) (F–F’’) and cup-5 (bp510) (G–G’’) expressing SCAV-3::GFP and NUC-1::pHTomato controlled by the heat-shock (hs) promoter. NUC-1::pHTomato is delivered to lysosomes labeled by SCAV-3::GFP at 24 hr post heat-shock treatment. (H–J) Confocal fluorescence images of the hypodermis in wild type (WT; H), daf-2(e1370) (I) and cup-5 (bp510) (J) expressing NUC-1::sfGFP::CHERRY controlled by the heat-shock (hs) promoter. (K) The average intensity of CHERRY in each lysosome was quantified. At least 20 animals were scored in each strain. In (C, D, K), data are shown as mean ± SD. Paired t testing (C, D) or one-way ANOVA with Tukey's multiple comparisons test (K) was performed to compare all other datasets with wild type at day 1 (C, D) or with wild type (K). **p<0.001. All other points had p>0.05. Scale bars: 5 μm.