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. Author manuscript; available in PMC: 2023 Jun 19.
Published in final edited form as: Nat Metab. 2022 Dec 19;4(12):1792–1811. doi: 10.1038/s42255-022-00706-6

Figure 6: TG and other lipid species accumulate independently of autophagy following mTORC1 inhibition.

Figure 6:

(A,B) Immunoblot (A) and TGs measured by pulse-chase with [1-14C]-oleate tracer (B) in Atg5+/+ and −/− MEFs treated for 16 hrs with vehicle, torin1 (250 nM), and/or BafA1 (250 nM), graphed as mean ± SD relative to vehicle-treated cells, n=3.

(C,D) Immunoblot (C) and TGs (D) measured by enzyme assay and normalized to protein content in parental and ATG7 KO HEK-293T cells treated as in (A,B), graphed as mean ± SD relative to vehicle-treated cells, n=3.

(E,F) Acyl-carnitine (E) and lysophospholipid (F) species measured by mass spectrometry in Atg5+/+ and −/− MEFs treated for 16 hrs with vehicle or torin1 (250 nM), graphed as mean ± SD relative to vehicle-treated cells, n=3.

(G) Immunoblot for autophagy markers in parental Tsc2−/− MEFs and sgUvrag and sgAtg14 clones following 4 hrs treatment with vehicle, rapamycin (20 nM), or torin1 (250 nM).

(H) TG accumulation measured by pulse-chase with [1-14C]-oleate tracer in the cells treated as in (G) for 16 hrs, graphed as mean ± SD relative to vehicle-treated cells, n=3.

(I) TG accumulation in parental Tsc2−/− MEFs and sgAtg14 clone following 16 hrs in amino acid-replete or free medium, measured and graphed as in (H) relative to amino acid-replete cells, n=3.

Statistical analysis by two-way ANOVA (B,D,E,F,H,I). n.s., not significant. * indicates P < .0001.