![Loss of VPS13C impairs phospho-Rab10-mediated lysosomal stress response. (A) Representative live-cell confocal images showing localization of VPS13C (VPS13C^mClover3, green) on the lysosomal membrane (LAMP1-RFP, magenta) and at lysosome-ER contact sites (LAMP1-RFP and mCherry-ER [magenta] or BFP-KDEL [blue]). Yellow arrows indicate co-localization. N = 3, from n ≥ 20 cells. Scale bar: 1 µm. (B) Quantification of the number of VPS13C-positive vesicles with or without overexpression of Rab10 WT, Rab10 T73E, and Rab10 T73A (N = 3, from n > 10 cells). (C) Quantification of the number of Rab10-positive vesicles with and without overexpression of VPS13C (N = 3, from n > 10 cells). VPS13C and Rab10 do not regulate each other’s recruitment. (D) Schematic of the isolation and enrichment of lysosomes from HEK-293 FT cells expressing LAMP1-RFP-3xHA. Pulldown of lysosomes via anti-HA-magnetic beads. Schematic was generated with http://BioRender.com. (E) Representative immunoblot of protein markers for cytosol, ER, mitochondria, and peroxisomes in enriched lysosomal fractions. (F and G) Quantification of relative enrichment of lysosomal markers compared with other organelle markers from LysoIP fractions showing the purity in lysosomal fractions (N = 3). (H) Quantification of total Rab10 protein levels from lysosomal fractions (N = 4). (I) Representative immunoblot from LysoIP after CQ treatment (100 µM, 16 h) showing lysosomal enrichment. (J) Representative immunoblots of phospho-Rab10 (pRab10) and total Rab10 protein levels from LysoIP after CQ treatment. (K) Quantification of lysosomal phospho-Rab10 protein levels (N = 4). The effect of VPS13C KD on lysosomal phospho-Rab10 under CQ-induced lysosomal stress is comparable with that under basal conditions. Data represented as mean ± SEM; one-way ANOVA with Tukey multiple comparison test (B), unpaired two-tailed t test (C and H), multiple unpaired t test (G), one-sample t test (K); ns: not significant (B, C, G, and H), *P < 0.05 (K). Source data are available for this figure: SourceData FS3.](https://cdn.rupress.org/rup/content_public/journal/jcb/223/5/10.1083_jcb.202304042/1/jcb_202304042_figs3.png?Expires=1768900016&Signature=n05XVuTxrb05VRA9ZHOOnxQCmYohse82zXoZO3Btk0Ie~6NhsNZyYg1nCVCBhK5KkoR7jC8Qd6ikGAo2LGAX2iv0iLwlpmz3m4EdQoFklEwMjvvKDftibbbWtXmxljNETIPJhBR71uI4ULZYpU8ZuKw8Mmw--wjESdjXrwO4aGU1HeogSGY1bhnJik932JfGxfIsKbTjBqDdZZvrp0SDd2MqGfF8E3Q7zmw1ENxZ6-2wVkYnzZtvI50xAW6Eyl4jzhUMhBUo83vGj-aM9zNKsiQvrL~g~GEqJq27WzG7fHk20Nfzf4RLqj7c25VteDxSpEN135Hm69jQG9krzdRAKw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Loss of VPS13C impairs phospho-Rab10-mediated lysosomal stress response. (A) Representative live-cell confocal images showing localization of VPS13C (VPS13C^mClover3, green) on the lysosomal membrane (LAMP1-RFP, magenta) and at lysosome-ER contact sites (LAMP1-RFP and mCherry-ER [magenta] or BFP-KDEL [blue]). Yellow arrows indicate co-localization. N = 3, from n ≥ 20 cells. Scale bar: 1 µm. (B) Quantification of the number of VPS13C-positive vesicles with or without overexpression of Rab10 WT, Rab10 T73E, and Rab10 T73A (N = 3, from n > 10 cells). (C) Quantification of the number of Rab10-positive vesicles with and without overexpression of VPS13C (N = 3, from n > 10 cells). VPS13C and Rab10 do not regulate each other’s recruitment. (D) Schematic of the isolation and enrichment of lysosomes from HEK-293 FT cells expressing LAMP1-RFP-3xHA. Pulldown of lysosomes via anti-HA-magnetic beads. Schematic was generated with http://BioRender.com. (E) Representative immunoblot of protein markers for cytosol, ER, mitochondria, and peroxisomes in enriched lysosomal fractions. (F and G) Quantification of relative enrichment of lysosomal markers compared with other organelle markers from LysoIP fractions showing the purity in lysosomal fractions (N = 3). (H) Quantification of total Rab10 protein levels from lysosomal fractions (N = 4). (I) Representative immunoblot from LysoIP after CQ treatment (100 µM, 16 h) showing lysosomal enrichment. (J) Representative immunoblots of phospho-Rab10 (pRab10) and total Rab10 protein levels from LysoIP after CQ treatment. (K) Quantification of lysosomal phospho-Rab10 protein levels (N = 4). The effect of VPS13C KD on lysosomal phospho-Rab10 under CQ-induced lysosomal stress is comparable with that under basal conditions. Data represented as mean ± SEM; one-way ANOVA with Tukey multiple comparison test (B), unpaired two-tailed t test (C and H), multiple unpaired t test (G), one-sample t test (K); ns: not significant (B, C, G, and H), *P < 0.05 (K). Source data are available for this figure: SourceData FS3.