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. 2013 May 15;27(10):1115–1131. doi: 10.1101/gad.198630.112

Figure 3.

Figure 3.

Unsaturated fatty acids rescue Tsc2−/− cell death under tumor-like stress. (A) The ability of unsaturated fatty acids to rescue Tsc2−/−, p53−/− cell death under tumor-like stress was assessed by culturing MEFs under SO conditions for 48 h with or without 50 μM oleic/linoleic acid or palmitic acid (P < 0.001). (B) Tsc2−/−, p53−/− MEFs were cultured for 48 h under Ored conditions in the presence and absence of 50 μM oleic/linoleic or palmitic acid (P < 0.001). (C) The ability of 35 μM oleic, palmitic, hexanoic, or octanoic acid to rescue Tsc2−/− cell death under 48 h of SO stress was determined by flow cytometry. (**) P < 0.005; (*) P < 0.001. (D) The ability of 50 μM oleic acid or oleic/linoleic acid to rescue Tsc2−/− cell death after 48 h of SOG stress was determined by flow cytometry. (**) P < 0.005; (*) P < 0.001. (E) mTORC1 and AKT signaling was assessed in response to addition of oleic or palmitic acid under lipid-deficient conditions. mTORC1 and AKT signaling in Tsc2+/+, p53−/− and Tsc2−/−, p53−/− MEFs was analyzed by blotting for the phosphorylation status of S6K1, S6, 4E-BP1, and AKT. (F) Scd1 mRNA levels in Tsc2+/+, p53−/− and Tsc2−/−, p53−/− MEFs exposed to 21% or 0.5% O2 for 24 h in replete, S, or SG medium were determined by qRT–PCR. (G) Tsc2+/+, p53−/− and Tsc2−/−, p53−/− MEFs were grown under 21% or 0.5% O2 in medium containing 10 mM [U-13C6]glucose or 3 mM [5-13C]glutamine for 24 h, and the relative contribution of glucose, glutamine, and serum-derived lipids to lipid synthesis in Tsc2−/− MEFs was determined from the NMR chemical spectra (see also Supplemental Fig. S3A,B). These results are displayed in histogram form (P < 0.005). (H) The relative levels of de novo unsaturated fatty acids in Tsc2+/+, p53−/− and Tsc2−/−, p53−/− MEFs cultured under normoxic (21% O2) and hypoxic (0.5% O2) conditions were calculated from the NMR chemical spectra and are presented as a bar graph (P < 0.001). (I) The relative levels of newly synthesized stearic, oleic, and linoleic acid in Tsc2−/−, p53−/− MEFs cultured under S and SO conditions as well as under SO conditions were assessed by GC-MS.