VPS45 KO cells fail to efficiently process soluble cargos and show a delayed maturation of cathepsin D. Control and VPS45 KO HeLa clones were incubated with various fluorescent substrates for indicated time periods (A-B: DQ OVA; C: DQ Green BSA; D: pHrodo Green Dextran). (A) Representative confocal microscopy images of DQ OVA processing in control and VPS45 KO HeLa clones are shown. After incubation with DQ OVA for 90 minutes, cells were fixed and stained for EEA1 and LAMP2. DAPI was used to visualize the nucleus. Enlarged images represent magnified views of boxed areas (8 µm × 8 µm). One representative experiment of 2 independent experiments is shown. Scale bars, 10 µm. (B-D) The rate of substrate processing was determined by flow cytometry as a change in MFI from baseline (fluorescence at 4°C). (B) Control and VPS45 KO cells were additionally treated with 25 µM CQ. Data are pooled from 4 independent experiments. (C) Data are pooled from 3 independent experiments. (D) Data are pooled from 4 independent experiments. (B-D) Error bars indicate mean ± SD. Statistical analysis was performed using 2-way ANOVA followed by Tukey or Sidak multiple comparisons test. *P < .05; ***P < 0,001; ****P < .0001. (E) Expression levels of immature (50 kDa), intermediate (46 kDa), and mature (28 kDa) cathepsin D were analyzed in control and VPS45 KO HeLa clones (n = 3 different clones per genotype) by immunoblotting. One representative experiment of 3 independent experiments is shown.