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. 2022 Aug 17;13:4848. doi: 10.1038/s41467-022-32515-6

Fig. 2. mTORC1 inactivation increases lysosomal protein catabolism through lysosomal acidification.

Fig. 2

a Cresyl violet accumulation in MEFs after 1 h ± torin 1 [400 nM]. Scale bars = 50 µm. b Quantification of cresyl violet accumulation in cells treated as in a; data are normalized replicate mean ± SEM (closed circles) and fields of view (open circles; ≥8 per replicate). c Lysosomal pH of MEFs after 1 h rapamycin [100 nM], torin 1 [250 nM], or AZD8055 [250 nM], quantified by lysosensor. d Lysosomal pH of MEFs after 1 h amino acid starvation (−aa), or 1 h aa starvation + 30 min aa restimulation (+aa), quantified by lysosensor. e Lysosomal pH, quantified by lysosensor, and degradation of lysosomally preloaded DQ BSA in MEFs after 1 h torin 1 at indicated concentrations. Measurements were conducted separately under identical conditions. f Lysosomal pH, quantified by lysosensor, in MEFs after 1 h torin 1 [400 nM] and bafilomycin A1 at indicated concentrations. The dashed line indicates mean lysosomal pH of control cells. g Degradation of lysosomally preloaded DQ BSA in MEFs after 1 h torin 1 [400 nM] and bafilomycin A1 at indicated concentrations. The dashed line indicates mean DQ BSA fluorescence of control cells. h Degradation of lysosomally preloaded DQ BSA and accumulation of cresyl violet (CV) in human carcinoma cell lines (MIA PaCa-2, A549) and fibroblasts (MRC-5) after 3 h ± torin 1 [400 nM]. Scale bars = 20 µm. i Quantification of DQ BSA fluorescence of cell lines shown in h. j Lysosomal pH in cell lines as in h after 1 h ± torin 1, quantified by lysosensor. b n = 5 biologically independent experiments; p-value was calculated using a two-tailed unpaired t-test with Welch correction. c–f, j Lysosensor data are mean ± SD (3 technical replicates). e, g, i DQ BSA data are mean ± SD (8–10 fields of view). c–j One representative of n = 3 biologically independent experiments. Source data are provided as a Source data file.