Figure 6.
USP45 depletion–induced F-actin patch formation is required for V-ATPase translocation to lysosomes. (A) Confocal microscopy analysis of F-actin structures stained with phalloidin in control (scr) and USP45 knockdown HeLa cells. Arrows indicate cytoplasmic F-actin patches. Scale bar, 20 μm. (B) Quantification of the area of cytoplasmic F-actin patches in A; data shown as mean ± SEM, n = 3, ≥ 35 cells. (C) Immunofluorescence analysis of colocalization of the F-actin patches and endolysosomal markers (EEA1, Rab7, and LAMP1) in control and USP45 knockdown cells with indicated antibodies. The scale bars showed 20 μm (original) and insets with 1.5x zoom. (D) Quantification of the colocalization of F-actin patches and endolysosomal markers in C. Pearson’s correlation coefficient was analyzed by ImageJ. Data are shown as mean ± SEM, n = 3, ≥ 30 cells. (E) Immunofluorescence analysis of colocalization of V-ATPase (V1H) and lysosome (LAMP1) in control and USP45 knockdown HeLa cells treated with LatA (200 nM) or Arp2/3 inhibitor CK666 (200 μM) for 2 h. Scale bar, 10 μm (original) and insets, 2.5x zoom. (F) Quantification of the colocalization of V1H and LAMP1 in E. Pearson’s correlation coefficient and colocalization rate were analyzed by ImageJ. Data are shown as mean ± SEM, n = 3, ≥ 25 cells. (G) Immunofluorescence analysis of colocalization of V-ATPase (V1H) and lysosome (LAMP1) in control and USP45 knockdown cells transfected with control (siCtrl) siRNA or siRNA targeting WASP family genes (siN-WASP and siWASH). Scale bar, 10 μm (original) and insets, 2.5x zoom. (H) Quantification of the colocalization of V1H and LAMP1 in G. Data are shown as mean ± SEM, n = 3, ≥ 25 cells. Significance was determined using one-way ANOVA and Dunnett’s (B) or Tukey’s (F and H) multiple comparisons test, and Student’s t test (D); ***P < 0.001; ****P < 0.0001; NS not significant.

USP45 depletion–induced F-actin patch formation is required for V-ATPase translocation to lysosomes. (A) Confocal microscopy analysis of F-actin structures stained with phalloidin in control (scr) and USP45 knockdown HeLa cells. Arrows indicate cytoplasmic F-actin patches. Scale bar, 20 μm. (B) Quantification of the area of cytoplasmic F-actin patches in A; data shown as mean ± SEM, n = 3, ≥ 35 cells. (C) Immunofluorescence analysis of colocalization of the F-actin patches and endolysosomal markers (EEA1, Rab7, and LAMP1) in control and USP45 knockdown cells with indicated antibodies. The scale bars showed 20 μm (original) and insets with 1.5x zoom. (D) Quantification of the colocalization of F-actin patches and endolysosomal markers in C. Pearson’s correlation coefficient was analyzed by ImageJ. Data are shown as mean ± SEM, n = 3, ≥ 30 cells. (E) Immunofluorescence analysis of colocalization of V-ATPase (V1H) and lysosome (LAMP1) in control and USP45 knockdown HeLa cells treated with LatA (200 nM) or Arp2/3 inhibitor CK666 (200 μM) for 2 h. Scale bar, 10 μm (original) and insets, 2.5x zoom. (F) Quantification of the colocalization of V1H and LAMP1 in E. Pearson’s correlation coefficient and colocalization rate were analyzed by ImageJ. Data are shown as mean ± SEM, n = 3, ≥ 25 cells. (G) Immunofluorescence analysis of colocalization of V-ATPase (V1H) and lysosome (LAMP1) in control and USP45 knockdown cells transfected with control (siCtrl) siRNA or siRNA targeting WASP family genes (siN-WASP and siWASH). Scale bar, 10 μm (original) and insets, 2.5x zoom. (H) Quantification of the colocalization of V1H and LAMP1 in G. Data are shown as mean ± SEM, n = 3, ≥ 25 cells. Significance was determined using one-way ANOVA and Dunnett’s (B) or Tukey’s (F and H) multiple comparisons test, and Student’s t test (D); ***P < 0.001; ****P < 0.0001; NS not significant.

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