Extended Data Fig. 4.

(A) ¹⁴C-TG turnover in PC3 cells treated with vehicle, torin1, or ATGLi, measured as in Figure 4C. ¹⁴C-TG abundance is graphed over time as mean ± SD relative to 0 hrs, n=3.

(B) Immunoblots for ATGL and DGAT1 levels from two separate experiments in cell lines treated with vehicle, rapamycin, or torin1 for 16 hrs. NS denotes a non-specific band.

(C) ¹⁴C-Fatty acid levels in Tsc2^−/− MEFs labeled with [1-¹⁴C]-palmitate, treated with vehicle, rapamycin, or torin1 for 16 hrs, graphed as mean ± SD relative to vehicle, n=3.

(D) Acyl-carnitine species in Tsc2^−/− MEFs in the experiment in Figure 1D, graphed as mean ± SD relative to vehicle, n=3.

(E,F) Acyl-carnitine species detected in mouse embryonic stem (ES) cells (E) and wild type, primary MEFs (F) in the experiments in Figures 1H,J, graphed as mean ± SD relative to vehicle, n=3.

(G) Representative thin layer chromatogram for Tsc2^−/− MEFs traced with [methyl-¹⁴C]-choline and treated with vehicle, rapamycin, or torin1 for 16 hrs, developed to separate phospholipids. PC, phosphatidylcholine; SM, sphingomyelin; LPC, lysophosphatidylcholine.

(H) Ratio of lysophosphatidylcholine to phosphatidylcholine in PC3 cells pulse-labeled with [methyl-¹⁴C]-choline as in (G); mean ± SD relative to vehicle, n=3.

(I,J) Glycerophosphocholine levels in HEK-293T (I) and PC3 (J) cells treated for 16 hrs with vehicle, rapamycin, or torin1, graphed as mean ± SD relative to vehicle, n=3.

(K,L) ¹⁴C-TG (K) and ¹⁴C-fatty acids (L) in PC3 cells measured as in Figures 4I,J and graphed as mean ± SD, n=3.

(M) Fatty acid accumulation in Tsc2^−/− MEFs labeled with [1-¹⁴C]-oleate following vehicle (DMSO) treatment with 1 hr pre-treatment with DGAT inhibitors and/or etomoxir; mean ± SD relative to 0 hrs, n=3.

(N) Triglyceride ion counts in the experiment shown in Figures 4L,M; mean ± SD relative to vehicle, n=3.

(O) Carnitine palmitoyl-transferase (CPT) gene expression in MEFs treated for 16 hrs with inhibitors, graphed as mean ± SD relative to vehicle, n=6.

Statistics: by one-way ANOVA (C,H-J,N), two-way ANOVA (D-F,K-M,O), and extra sum-of-squares F-test (A). n.s., not significant.