dUsp45 overexpression impairs lysosomal acidification and V-ATPase lysosomal localization. (A) Confocal microscopy analysis showed colocalization of mCherry-Atg8a and GFP-LAMP1 in control or dUsp45^WT expressing wL3 larval fat body cells. Scale bar, 20 μm. (B) Quantification of the colocalization of Atg8a and LAMP1 in A. Pearson’s correlation coefficient was analyzed by ImageJ. Data are shown as mean ± SEM, n = 3, ≥ 20 cells. (C) Confocal microscopy analysis of wL3 larval fat body cells expressing Luc (Ctrl), dUsp45^WT, or dUsp45^RNAi with Cg-Gal4 driver and stained with the fluorescent dye LysoTracker Red. Scale bar, 20 μm. (D) Quantification of the number of LysoTracker-positive dots per cell in C; data shown as mean ± SEM, n = 3, ≥ 20 cells. (E) Quantification of the number of MagicRed puncta per cell in dUsp45 knockdown wL3 larval fat body cells. Data are shown as mean ± SEM, n = 3, ≥ 15 cells. (F) Western blot analysis of Cathepsin-L (CTHL) expression levels in the larval fat bodies expressing Luc (Ctrl) or dUsp45^RNAi under the control of Cg-Gal4. (G) Quantification of Cathepsin-L expression normalized to Actin in F. Data shown as mean ± SEM of four independent experiments. (H) Confocal microscopy analysis of localization of VhaSFD (GFP positive) and mCherry-Atg8a in control and dUsp45^WT overexpression wL3 larval fat body cells. The arrows showed the colocalized signals of VhaSFD and Atg8a. The scale bars showed 20 μm (original) and 10 μm (zoom-in). (I) Line-scan profiles of fluorescence intensity for mCherry-Atg8a and GFP-VhaSFD along the white line in H. Significance was determined using one-way ANOVA and Dunnett’s multiple comparisons test (D and G), and Student’s t test (B and E); *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: SourceData F2.