CORO1B knockdown promotes lysosomal function and V-ATPase translocation to lysosome in an N-WASP-dependent manner. (A) Immunofluorescent analysis of LysoTracker (LTK) in control and CORO1B knockdown HeLa cell. Scale bar, 10 μm. (B) Quantification of LTK dot number in A. Data are shown as mean ± SEM in n = 3, ≥30 cells. (C) Confocal microscopic analysis of DQ-BSA in control and CORO1B knockdown cells. Scale bar, 10 μm. (D) Quantification of DQ-BSA number in C. Data are shown as mean ± SEM in n = 3 , ≥30 cells. (E) Confocal microscopic analysis of LTK signals in control and USP45 knockdown cells with or without Flag-Coro1B overexpression. Scale bar, 10 μm. (F) Quantification of LTK dot number in E. Data shown as mean ± SEM in n = 3, ≥20 cells. (G) Lysosomal immunoprecipitation (LysoIP) analysis of V-ATPase subunit (V1H) in enriched lysosome lysate of control and CORO1B knockdown HEK293T cells. TMEM192-Flag was emerged as negative control. (H) Quantification of V1H normalized to HA in G. Data shown as mean ± SEM in three of independent experiments. (I) Immunofluorescent analysis of the colocalization of the V-ATPase subunit (V1H) and lysosome marker (LAMP1) in control and Coro1B-depleted HeLa cells with knockdown of control or WASP family proteins (N-WASP, WASH) by the indicated siRNAs. (J) Quantification of colocalization of V1H and LAMP1 in I. Data are shown as mean ± SEM in n = 3, ≥20 cells. Significance was determined using one-way ANOVA and Dunnett’s (B, D, and H) or Tukey‘s (F and J) multiple comparisons test; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData FS5.