Figure S1.
dUsp45 impairs autophagy and V-ATPase translocation to the autolysosome. (A) Confocal microscopic analysis of mCherry-Atg8a puncta in control (Luc), dUsp45 knockdown, and dUsp45 rescue (dUsp45 depletion with dUsp45WT over-expression) larvae fat body. Scale bar, 20 μm. (B) Quantification of Atg8a puncta number in A. Data are shown as mean ± SEM in n = 3, ≥30 cells. (C) Clonal analysis of wL3 larval fat body cells showing mCherry-Atg8 signals in control and dUsp45 knockdown cells (marked with NLS-GFP). Scale bar, 20 μm. (D) Quantification of Atg8 puncta size in control and dUsp45 knockdown cell shown in C. Data shown as mean ± SEM, n = 3, ≥ 20 cells. (E) Western blot analysis of expression level of wild-type (WT) and catalytic mutant (C315A) of dUsp45 in larvae fat body indicated by Flag antibody. (F) Representative TEM images showed the ultrastructure of control (Luciferase), dUsp45 wild-type (WT), and catalytic mutant (CA) overexpression wL3 larval fat body cells. Black arrow indicates autolysosome. Scale bar, 1 μm. (G) Quantification of autolysosome size in F; data shown as mean ± SEM, n = 3, ≥ 20 autolysosomes. (H) Western blot analysis of Ref2P expression in wandering larvae fat body with control and dUsp45WT overexpression. (I) Quantification of Ref2P was normalized to Tubulin. Data are shown as mean ± SEM in four of independent experiments. (J) Diagram shows the percentage of control, dUsp45 overexpression, and knockdown larvae pupariated at indicated days. The data were collected from ≥90 flies in each group. (K) Confocal microscopy analysis of Vha13 (GFP-positive) and mCherry-Atg8a localization in control and dUsp45WT-overexpressing wL3 larval fat body cells. Scale bar, 20 μm (original) and 10 μm (zoom-in). (L) Line-scan profiles of fluorescence intensity for mCherry-Atg8a and GFP-Vha13 along the white line in K. Significance was determined using one-way ANOVA and Tukey‘s (B) or Dunnett’s (G) multiple comparisons test, and Student’s t test (D and I); *P < 0.05; ****P < 0.0001. Source data are available for this figure: SourceData FS1.

dUsp45 impairs autophagy and V-ATPase translocation to the autolysosome. (A) Confocal microscopic analysis of mCherry-Atg8a puncta in control (Luc), dUsp45 knockdown, and dUsp45 rescue (dUsp45 depletion with dUsp45WT over-expression) larvae fat body. Scale bar, 20 μm. (B) Quantification of Atg8a puncta number in A. Data are shown as mean ± SEM in n = 3, ≥30 cells. (C) Clonal analysis of wL3 larval fat body cells showing mCherry-Atg8 signals in control and dUsp45 knockdown cells (marked with NLS-GFP). Scale bar, 20 μm. (D) Quantification of Atg8 puncta size in control and dUsp45 knockdown cell shown in C. Data shown as mean ± SEM, n = 3, ≥ 20 cells. (E) Western blot analysis of expression level of wild-type (WT) and catalytic mutant (C315A) of dUsp45 in larvae fat body indicated by Flag antibody. (F) Representative TEM images showed the ultrastructure of control (Luciferase), dUsp45 wild-type (WT), and catalytic mutant (CA) overexpression wL3 larval fat body cells. Black arrow indicates autolysosome. Scale bar, 1 μm. (G) Quantification of autolysosome size in F; data shown as mean ± SEM, n = 3, ≥ 20 autolysosomes. (H) Western blot analysis of Ref2P expression in wandering larvae fat body with control and dUsp45WT overexpression. (I) Quantification of Ref2P was normalized to Tubulin. Data are shown as mean ± SEM in four of independent experiments. (J) Diagram shows the percentage of control, dUsp45 overexpression, and knockdown larvae pupariated at indicated days. The data were collected from ≥90 flies in each group. (K) Confocal microscopy analysis of Vha13 (GFP-positive) and mCherry-Atg8a localization in control and dUsp45WT-overexpressing wL3 larval fat body cells. Scale bar, 20 μm (original) and 10 μm (zoom-in). (L) Line-scan profiles of fluorescence intensity for mCherry-Atg8a and GFP-Vha13 along the white line in K. Significance was determined using one-way ANOVA and Tukey‘s (B) or Dunnett’s (G) multiple comparisons test, and Student’s t test (D and I); *P < 0.05; ****P < 0.0001. Source data are available for this figure: SourceData FS1.

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