Loss of NDST3 leads to lysosomal hyperacidification and dysfunction.Figure 2(A) Determination of lysosomal pH with the ratiometric probe LysoSensor Yellow/Blue dextran. WT and NDST3 KO RPE1 cells were stained with LysoSensor Yellow/Blue dextran and then observed for fluorescence. Yellow fluorescence represents more acidic lysosomal environment, and blue fluorescence represents more neutral lysosomal environment. (B) The calibration curve for the lysosome pH. RPE1 cells were incubated with a series of pH calibration buffers (pH 4.0, 4.5, 5.0, 5.5 and 6.0) in the presence of 10 μM monensin and 30 μM nigericin and examined for the fluorescence ratio of LysoSensor Yellow/Blue dextran staining by a plate reader. (C) Lysosomal pH values in WT and NDST3 KO RPE1 cells calculated from the fluorescence ratio of LysoSensor Yellow/Blue dextran staining against the pH calibration curve in (B) (n = 3 independent cultures, *P = 0.0242). (D) Lysosomal pH values measured for WT and NDST3 KO RPE1 cells after the treatment with Baf A1 (100 nM, 1h) (n = 3 independent cultures, *P = 0.0305). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm. (E) SDS-PAGE and immunoblotting analysis of the maturation of Cathepsin B (Cat B) in the lysosomes isolated from WT and NDST3 KO RPE1 cells. The ratio of mature Cat B (heavy chain) to pro-cathepsin B (ProCat B) was calculated to evaluate the maturation of Cat B (n = 3 independent experiments, ***P = 0.0007). LAMP1 was set as a loading control. Data information: Error bars represent ± standard deviation. Scale bar, 10 μm. (F) Magic Red assay for Cat B proteolytic activity. WT and NDST3 KO RPE1 cells were treated with vehicle (VEH) or Baf A1 (100 nM, 1 h) and then stained with MR-(RR)2 to measure the Cathepsin B activity (n = 48 cells in the VEH-treated WT group, n = 37 cells in the VEH-treated NDST3 KO group, n = 64 cells in the Baf A1-treated WT group, n = 42 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001). (G) Alexa Fluor (AF) 488-dextran degradation assay. WT and NDST3 KO RPE1 cells were loaded with AF488-dextran in culture medium and observed for fluorescence. The AF488 fluorescence signals without a chase period, with a 4-h chase period, and with the 4-h chase period in the presence of Baf A1 (100 nM, 1h) were recorded as fluorescence of uptake, chase, and chase with Baf A1, respectively. The AF488 intensity per cell was quantified and calculated into degradation percentage according to the equation in Materials and Methods (n = 62 cells in the VEH-treated WT group, n = 36 cells in the VEH-treated NDST3 KO group, n = 65 cells in the Baf A1-treated WT group, n = 78 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001). (H) Analysis of the conversion of LC3B-I to LC3B-II and the levels of p62. WT and NDST3 KO RPE1 cells were cultured with or without 100 nM rapamycin (Rapa) for 6 h and LC3B-I, LC3B-II, p62, and GAPDH levels in the total cell lysates were determined by SDS-PAGE and immunoblotting. The ratios of LC3B-II to LC3B-I (n = 3 independent experiments, *P = 0.0451, **P = 0.0042) and the p62 levels normalized to β-tubulin (n = 3 independent experiments, *P(Rapa-) = 0.0158, *P(Rapa+) = 0.0183) are quantified. Data information: Error bars represent ± standard deviation. Scale bar, 10 μm. (I) The LC3 turnover assay. WT and NDST3 KO RPE1 cells were cultured in the presence of 100 nM Rapa and with or without 50 μM chloroquine (CQ) for 6 h, and LC3B-II levels in the total cell lysates were determined by immunoblotting. The fold change of LC3B-II levels was shown by comparing the samples with and without CQ treatment (n = 6 independent experiments, ***P = 0.0007). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm. (J) The LC3 turnover assay after a 48-h Baf A1 treatment. WT and NDST3 KO RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B-II fold change as in (I) (n = 6 independent experiments, **P = 0.0014). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.