Extended Data Fig. 7 : TG and other lipid species accumulate independently of autophagy following mTORC1 inhibition.
From: mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species
(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 or BafA1 (250 nM), graphed as mean ± SD relative to vehicle-treated cells, n = 3. (c,d) Immunoblot (c) and triglyceride levels measured by enzyme assay (D) and normalized to protein content from the same sample for parental, ATG7 knockout, and FIP200 knockout HEK-293T cells treated with vehicle or bafilomycin A1 (250 nM) for 16 hrs, graphed as mean ± SD relative to vehicle-treated cells, n = 3. (e, f) Immunoblot (e) and triglyceride levels (f) corresponding to the results shown in Fig. 6e, f. Triglyceride levels are graphed as mean ± SD relative to vehicle-treated cells, n = 3. (g, h) Immunoblot (g) and triglyceride levels measured by enzyme assay (h) and normalized to protein content from the same sample for parental and FIP200 knockout HEK-293T cells treated with vehicle, torin1 (250 nM), and/or bafilomycin A1 (250 nM) for 16 hrs, graphed as mean ± SD relative to vehicle-treated cells, n = 3. (i) EGFR degradation assay in parental Tsc2−/− MEFs and sgUvrag or sgAtg14 clones. Cells were serum-starved for 16 hrs and stimulated with EGF (10 ng/mL) for the indicated times. (j) Immunoblot for autophagy markers in parental Tsc2−/− MEFs and sgAtg14 clone after 4 hrs in amino acid replete or free medium. Statistical analysis by two-way ANOVA (B,D,F,H). n.s., not significant.
