Figure 1.
dUsp45 depletion enhances autophagosome formation and autophagic flux. (A and B) The clonal knockdown of dUsp45 (GFP positive) in the larval fat bodies using the flp-out system resulted in an elevated presence of mCherry-Atg8a puncta, compared with controls (GFP negative). Secondary instar larvae (L2) were incubated in normal food (A) or with chloroquine (CQ, 10 mg/ml) for 6 h (B). Knockdown clones are indicated by the dashed line. Scale bar: 20 μm. (C) Quantification results of the number and size of Atg8a puncta were depicted for conditions (A and B). Data are shown as mean ± SEM, n = 3, ≥ 30 cells. (D) Confocal microscopy analysis of autophagic flux, as determined by the expression of GFP-mCherry-Atg8a with the fat body-specific Cg-Gal4 driver, in control (Ctrl) and dUsp45 knockdown larvae. Scale bar, 20 μm. (E) Quantification of total Atg8a puncta and ratio of autolysosomes (GFP− mCherry+) to total Atg8a puncta per cell in D; data are shown as mean ± SEM, n = 3, ≥ 30 cells. (F) RT-PCR analysis of dUsp45 mRNA levels in the fat body of second instar (L2) larvae and wandering third-instar (wL3) larvae. Quantification results indicated dUsp45 expression levels normalized to Actin. (G) The clonal expression of dUsp45-WT (GFP positive), but not catalytic mutant dUsp45-C315A (GFP positive), in the wL3 larval fat bodies using the flp-out system resulted in an enlarged size of Atg8a puncta, compared with controls (GFP negative). The clones were indicated by the dashed line. Scale bar: 20 μm. (H) Quantification of the number and size of Atg8a puncta per cell in G; data shown as mean ± SEM, n = 3, ≥ 25 cells. (I) The clonal expression of dUsp45-WT, but not catalytic mutant dUsp45-C315A, in the wL3 larval fat bodies resulted in increased Ref2P signal, compared with controls. The clones were indicated by white lines. Scale bar: 20 μm (original) and 10 μm (zoom-in). (J) Quantification of Ref2P intensity per cell and percentage of the Atg8a puncta colocalized with Ref2P dots. Data are shown as mean ± SEM, n = 3, ≥ 25 cells. Significance was determined using one-way ANOVA and Dunnett’s multiple comparisons test (C, E, H, and J), and Student’s t test (F); *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: SourceData F1.

dUsp45 depletion enhances autophagosome formation and autophagic flux. (A and B) The clonal knockdown of dUsp45 (GFP positive) in the larval fat bodies using the flp-out system resulted in an elevated presence of mCherry-Atg8a puncta, compared with controls (GFP negative). Secondary instar larvae (L2) were incubated in normal food (A) or with chloroquine (CQ, 10 mg/ml) for 6 h (B). Knockdown clones are indicated by the dashed line. Scale bar: 20 μm. (C) Quantification results of the number and size of Atg8a puncta were depicted for conditions (A and B). Data are shown as mean ± SEM, n = 3, ≥ 30 cells. (D) Confocal microscopy analysis of autophagic flux, as determined by the expression of GFP-mCherry-Atg8a with the fat body-specific Cg-Gal4 driver, in control (Ctrl) and dUsp45 knockdown larvae. Scale bar, 20 μm. (E) Quantification of total Atg8a puncta and ratio of autolysosomes (GFP− mCherry+) to total Atg8a puncta per cell in D; data are shown as mean ± SEM, n = 3, ≥ 30 cells. (F) RT-PCR analysis of dUsp45 mRNA levels in the fat body of second instar (L2) larvae and wandering third-instar (wL3) larvae. Quantification results indicated dUsp45 expression levels normalized to Actin. (G) The clonal expression of dUsp45-WT (GFP positive), but not catalytic mutant dUsp45-C315A (GFP positive), in the wL3 larval fat bodies using the flp-out system resulted in an enlarged size of Atg8a puncta, compared with controls (GFP negative). The clones were indicated by the dashed line. Scale bar: 20 μm. (H) Quantification of the number and size of Atg8a puncta per cell in G; data shown as mean ± SEM, n = 3, ≥ 25 cells. (I) The clonal expression of dUsp45-WT, but not catalytic mutant dUsp45-C315A, in the wL3 larval fat bodies resulted in increased Ref2P signal, compared with controls. The clones were indicated by white lines. Scale bar: 20 μm (original) and 10 μm (zoom-in). (J) Quantification of Ref2P intensity per cell and percentage of the Atg8a puncta colocalized with Ref2P dots. Data are shown as mean ± SEM, n = 3, ≥ 25 cells. Significance was determined using one-way ANOVA and Dunnett’s multiple comparisons test (C, E, H, and J), and Student’s t test (F); *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: SourceData F1.

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