Figure 3.
Mammalian USP45 negatively regulates autophagy. (A) Schematic presentation of the domain structures of human USP45 and Drosophila dUsp45. (B) Immunofluorescence analysis of LC3 puncta in control (scr) or USP45 knockdown HeLa cells with or without bafilomycin A1 (BafA1) treatment. Scale bar, 20 μm. (C) Quantification of the number of LC3 puncta in control and USP45 knockdown cells in B; data shown as mean ± SEM, n = 3, ≥ 30 cells. (D) Western blot analysis of LC3 and p62 levels in control and USP45 depleted HeLa cells with or without BafA1 treatment. (E) Quantification of LC3 and p62 expression normalized to GAPDH in D. Data shown as mean ± SEM of four independent experiments. (F) Confocal microscopy analysis of autophagy flux in mRFP-EGFP-LC3 transfected control or USP45 knockdown HeLa cells. Scale bar showed 10 μm (original) and 5 μm (zoom-in). (G) Quantification of the number of total LC3 puncta and the ratio of autolysosomes (RFP+GFP−) to total LC3 puncta. Data as shown by mean ± SEM, n = 3, ≥25 cells/condition. (H) Western blot analysis of phospho-p70 S6 kinase (pS6K), total p70 S6 kinase (S6K), and mTOR expression levels in control and USP45 knockdown HeLa cells. (I) Quantification of phospho-S6K normalized to total S6K, and mTOR levels normalized to GAPDH in H. Data shown as mean ± SEM of three independent experiments. (J) Western blot analysis of ULK1, VPS34, and Beclin1 expression levels in control and USP45 knockdown HeLa cells. (K) Quantification of ULK1, VPS34, and Beclin1 levels normalized to GAPDH in J. Data shown as mean ± SEM of three independent experiments. (L) Immunofluorescent analysis of GFP-TFEB localization in control and USP45 knockdown HeLa cells. Scale bar, 20 μm. (M) Quantification of the percentage of cells with nuclear GFP-TFEB signal. Data are shown by mean ± SEM, n = 30 images, ≥5 cells/image. Significance was determined using one-way ANOVA and Dunnett’s multiple comparisons test (C, E, G, I, and K), and Student’s t test (M); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS not significant. Source data are available for this figure: SourceData F3.

Mammalian USP45 negatively regulates autophagy. (A) Schematic presentation of the domain structures of human USP45 and Drosophila dUsp45. (B) Immunofluorescence analysis of LC3 puncta in control (scr) or USP45 knockdown HeLa cells with or without bafilomycin A1 (BafA1) treatment. Scale bar, 20 μm. (C) Quantification of the number of LC3 puncta in control and USP45 knockdown cells in B; data shown as mean ± SEM, n = 3, ≥ 30 cells. (D) Western blot analysis of LC3 and p62 levels in control and USP45 depleted HeLa cells with or without BafA1 treatment. (E) Quantification of LC3 and p62 expression normalized to GAPDH in D. Data shown as mean ± SEM of four independent experiments. (F) Confocal microscopy analysis of autophagy flux in mRFP-EGFP-LC3 transfected control or USP45 knockdown HeLa cells. Scale bar showed 10 μm (original) and 5 μm (zoom-in). (G) Quantification of the number of total LC3 puncta and the ratio of autolysosomes (RFP+GFP−) to total LC3 puncta. Data as shown by mean ± SEM, n = 3, ≥25 cells/condition. (H) Western blot analysis of phospho-p70 S6 kinase (pS6K), total p70 S6 kinase (S6K), and mTOR expression levels in control and USP45 knockdown HeLa cells. (I) Quantification of phospho-S6K normalized to total S6K, and mTOR levels normalized to GAPDH in H. Data shown as mean ± SEM of three independent experiments. (J) Western blot analysis of ULK1, VPS34, and Beclin1 expression levels in control and USP45 knockdown HeLa cells. (K) Quantification of ULK1, VPS34, and Beclin1 levels normalized to GAPDH in J. Data shown as mean ± SEM of three independent experiments. (L) Immunofluorescent analysis of GFP-TFEB localization in control and USP45 knockdown HeLa cells. Scale bar, 20 μm. (M) Quantification of the percentage of cells with nuclear GFP-TFEB signal. Data are shown by mean ± SEM, n = 30 images, ≥5 cells/image. Significance was determined using one-way ANOVA and Dunnett’s multiple comparisons test (C, E, G, I, and K), and Student’s t test (M); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS not significant. Source data are available for this figure: SourceData F3.

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