Loss of VPS13C impairs lysosomal hydrolytic activity and acidification in dopaminergic neurons. (A–D) Representative immunoblots showing protein levels of mature cathepsin B (CTSB) (A) and mature cathepsin D (CTSD) (C) with relative protein quantifications, respectively (B and D) (N = 3). (E) Representative live-cell confocal images of Magic Red cathepsin B staining in control neurons showing stronger cathepsin B intensity (white arrow) and in VPS13C KD neurons showing lower cathepsin B intensity (yellow arrow). Scale bar: 10 µm, inset: 1 µm. Dashed line represents the outline of the cell. (F and G) Quantification of the number of Magic Red cathepsin B-positive puncta per cell area (F) and the mean fluorescence intensity of Magic Red cathepsin B-positive puncta (G) (N = 4, from n = 22 cells [control] and n = 19 cells [VPS13C KD]). (H) Representative live-cell confocal images of LysoTracker Red DND-99 staining in control neurons showing stronger LysoTracker intensity (white arrow) and in VPS13C KD neurons showing reduced LysoTracker intensity (yellow arrow). Scale bar: 10 µm, inset: 1 µm. Dashed line represents the outline of the cell. (I and J) Quantification of the number of LysoTracker Red–positive puncta per cell area (I) and the mean fluorescence intensity of LysoTracker Red-positive puncta (J) (N = 4, from n = 19 cells). (K) Schematic of lysosomal phenotypes in VPS13C deficiency in comparison with healthy control. VPS13C-deficient cells have enlarged lysosomes that tether together, are less motile, and have impaired lysosomal hydrolytic activity and acidification. Our data suggests that VPS13C regulates lysosomal homeostasis through the regulation of phospho-Rab10 on the lysosomal membrane. Data represented as mean ± SEM; unpaired two-tailed t test (B, D, F, G, I, and J); **P < 0.01 (B, D, and J), ****P < 0.0001 (F, G, and I). Source data are available for this figure: SourceData F7.