USP45 negatively regulates lysosomal activity by altering V-ATPase endolysosomal trafficking. (A) Western blot analysis of Cathepsin-L (CTHL) levels in control (scr) and USP45 knockdown HeLa cells. (B) Quantification of mature CTHL expression normalized to tubulin in A. Data shown as mean ± SEM of three independent experiments. (C) Immunofluorescence analysis of DQ-BSA puncta in control or USP45 knockdown HeLa cells. Scale bar, 20 μm. (D) Quantification of the number of DQ-BSA puncta in control and USP45 knockdown cells treated as C; data are shown as mean ± SEM, n = 3, ≥ 30 cells. (E) Confocal microscopy analysis of colocalization of the V-ATPase subunit (V0D) and endolysosomal markers (EEA1, Rab7, and LAMP2) in control and USP45 knockdown cells with indicated antibodies. The scale bars show 10 μm (original) and 4 μm (zoom-in). (F) Quantification of the colocalization of V0D and endolysosomal markers in E. Pearson’s correlation coefficient and colocalization rate were analyzed by ImageJ. Data are shown as mean ± SEM, n = 3, ≥ 30 cells. Significance was determined using Student’s t test (B, D, and F); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS not significant. Source data are available for this figure: SourceData F4.