1 INTRODUCTION
2 MATERIALS AND EQUIPMENT
2.1 Strains
2.2 Buffers
2.3 Equipment
2.4 Software
3 OVERVIEW OF THE EXPERIMENTAL DESIGN
Fig.1 Lysosome maturation assay. A, B Diagram of the assay to examine lysosome maturation in the hypodermis using heat-shock (HS) induction of NUC-1::sfGFP::CHERRY. C–J Merged confocal fluorescence images of the hypodermis in wild type (WT) and cup-5(bp510) expressing NUC-1::sfGFP::CHERRY at 4, 8, 12 and 24 h post HS. White arrowheads indicate structures labeled by CHERRY only and white arrows indicate overlap of GFP and CHERRY. K Overlap of GFP and CHERRY is quantified by Pearson’s correlation coefficient. At least five animals were scored at each time point in each strain. Data are shown as mean ± SD. Two-way ANOVA with Tukey’s multiple comparison test was used to compare mutant datasets with the wild type. *p < 0.05, ** p < 0.01; N.S.: no significance. Scale bars: 5 μm |
4 STEP-BY-STEP PROCEDURE
4.1 Lysosome acidification and maturation assay
4.2 Lysosome acidity assay
Fig.2 Lysosome acidity assay. A–C Confocal fluorescence images of the hypodermis at adult day 2 in wild type (WT), daf-2(e1370) and cup-5(bp510) expressing NUC-1::pHTomato controlled by the heat-shock (hs) promoter. Scale bars: 5 μm. D The average intensity of pHTomato per lysosome is quantified. At least 20 animals were scored in each strain. Data are shown as mean ± SD. The paired t-test was performed to compare the mutant datasets with the wild type. **p < 0.001 |
4.3 Lysosome degradation activity assay
4.3.1 Quantification of NUC-1::CHERRY cleavage
4.3.2 Examination and quantification of CPL-1 processing
Fig.3 Lysosome degradation activity assay. A, B Western blot analysis of NUC-1::CHERRY cleavage in wild type (WT), cup-5(bp510) and daf-2(e1370) at day 1 of adulthood. The percentage of cleaved CHERRY was quantified and is shown in the right panels. C, D Western blot analysis of CPL-1 processing in wild type (WT), cup-5(bp510) and daf-2(e1370) at day 1 of adulthood. The percentage of mature CPL-1 was quantified and is shown in the right panels. Three independent experiments were performed. Data are shown as mean ± SD. The paired t-test was performed to compare the mutant datasets with the wild type. *p < 0.05, ** p < 0.001 |