(a)Quantitation of Lysosomal Enrichment Score for p18, RagA, RagC and mTOR based on endogenous LAMP2 staining in U2OS cells that were starved for amino acids and glucose for 2h (‘-AA/G’), or starved for 1.5h and restimulated for amino acids and glucose for 30 min (‘+AA/G’) (mean ± S.D., N =16 cells/condition, *** p=0.0003, * p=0.0274, **** p<0.0001, two-sided unpaired t-tests). (b) Representative immunofluorescence images analyzed in (a). Scale bar 10 μm. (c) FRAP time-lapse of single lysosomes in U2OS cells expressing GFP-RagB + FLAG-RagC and subjected to the indicated nutrient conditions. “Compl. Media” indicates that cells were kept continuously in complete media, whereas “+AA/G” cells were starved of amino acids and glucose for 1.5hr then restimulated with amino acids and glucose for 30 min. “-AA/G/dFBS” indicates that cells were deprived of dialyzed FBS during starvation whereas -AA/G were starved in the presence of dialyzed FBS. Scale bar 1 μm. (d) Fluorescence over time curves from FRAP experiments in (c). Each curve is the average ± S.E.M. of [Compl. media N=30, +AA/G N=24, -AA/G/dFBS N=28, -AA/G N=27] lysosomes. (e) Plot showing inverse correlation between recovery fraction and halftime (t1/2) from the curves in (d). Shown are best fit values with 95% confidence intervals. (f) Fluorescence over time curves from FRAP experiments on SW780 Nprl2−/− cells and SW780 Nprl2 rescue (Nprl2res) cells expressing GFP-RagB that were either starved or restimulated for amino acids and glucose. Each curve is the average ± S.E.M. of [grey N=22, dark blue N=13, light blue N=20, red N=18] lysosomes. (g) Fluorescence over time curves from FRAP experiments on U2OS cells coexpressing GFP-RagBQ99L (nucleotide hydrolysis deficient mutant) or GFP-RagBWT along with FLAG-RagC, which were either starved or restimulated for amino acids and glucose. Each curve is the average ± S.E.M. of [grey N=20, dark blue N=16, light blue N=22, red N=21] lysosomes. See Supplementary Table 1 for statistics source data.