Figure 1.

VPS13C is a Rab7-binding protein implicated in maintaining lysosomal homeostasis. (A) Live HeLa cells expressing full-length VPS13C^Halo with WT mCherry-Rab7a (top row), constitutively active mCherry-Rab7a^Q67L (middle row), or mCherry-Rab7a^T22N (bottom row). (B) IB of VPS13C in WT HeLa cells and two individual clonal cell lines after CRISPR-Cas9–mediated KO of VPS13C. (C and D) IB of Lamp1 and Cathepsin D in WT and VPS13C^KO cells (C), quantified in D. α-Tubulin was used as a loading control. n = 3 biological replicates. (E and F) Live WT and VPS13C^KO HeLa cells stained with LysoTracker Red DND-99 (50 nM; E), quantified in F; n = 3 biological replicates, >182 cells per cell line. (G) IB of TFEB in WT, VPS13C^KO, and VPS13C^Rescue HeLa cells. As a positive control, WT cells were treated with 1 μM Torin-1 for 1 h. α-Tubulin was used as a loading control. Images from the same blot as Fig. S1 E. (H) Quantification of the ratio of unphosphorylated (lower band) to phosphorylated (upper band) TFEB from G. n = 3 biological replicates. Scale bars, 20 μm. Inset scale bars, 5 μm. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data were compared using two-sided t tests. Error bars represent ±SD. Source data associated with this figure can be found at https://doi.org/10.5281/zenodo.6416363. Source data are available for this figure: SourceData F1.