Figure 7: Induction of endosomal delivery to the lysosome is required for the changes in cellular lipid species following mTORC1 inhibition.

(A,B) Relative TG (A) and lysophospholipid (B) class sums measured by mass spectrometry in Tsc2^−/− MEFs treated for 16 hrs with vehicle, torin1 (250 nM), VPS34-IN1 (5 μM), and/or Dynasore (80 μM), graphed as mean ± SD relative to vehicle-treated cells, n=3.

(C) Fatty acid accumulation measured by pulse-chase with [1-¹⁴C]-oleate tracer in the presence of DGAT inhibitors (3 μM each) and etomoxir (20 μM) in Tsc2^−/− MEFs over a time course of vehicle or torin1 (250 nM) treatment, following 1 hr pre-treatment with endocytosis inhibitors (5 μM VPS34-IN1, 1 μM PIK-FYVEi, 80 μM Dynasore, or 20 μM PitStop 2). Graphed as mean ± SD relative to vehicle-treated cells at time 0, n=3.

(D,E) Endocytosis of transferrin (time course in D) and 10-kDa dextran (E) following 4 hrs of vehicle, rapamycin (20 nM), or torin1 (250 nM) treatment in Tsc2^−/− MEFs, graphed as mean ± SD, n=3.

(F,G) mTORC1 inhibition enhances hydrolysis of endocytosed BSA in Tsc2^−/− MEFs (F) and PC3 cells (G). Following 1 hr pretreatment with vehicle, rapamycin (20 nM) or torin1 (250 μM), cells were co-labeled for 3 hrs with BSA Alexa Fluor 647 (uptake, red in merged) and DQ-Green BSA (cleavage, green in merged) (10 μg/mL each). Nuclei are shown in blue.

(H-K) Quantification of the results from the experiments shown in (F) and (G). (H,J) Relative BSA Alexa Fluor 647 uptake into individual cells. (I,K) Relative DQ-Green-BSA fluorescence (cleavage) normalized to BSA Alexa Fluor 647 (uptake) on a per-cell basis. Graphed as mean ± SD relative to vehicle-treated cells, n=108-133 cells.

a.u., Arbitrary Units. Statistical analysis by two-way ANOVA (A-E), and one-way ANOVA (H-K). n.s., not significant. In (B) and (C) * indicates P < 0.001.