LOPMENTFigure 3. Unsaturated fatty acids rescue Tsc2??cell death below tumor-like tension. (A) The capacity of unsaturated fatty acids to rescue Tsc2?? p53??cell death below tumor-like strain was assessed by culturing MEFs under SO circumstances for 48 h with or with no 50 mM oleic/linoleic acid or palmitic acid (P 0.001). (B) Tsc2?? p53??MEFs were cultured for 48 h below Ored situations in the presence and absence of 50 mM oleic/linoleic or palmitic acid (P 0.001). (C) The capability of 35 mM oleic, palmitic, hexanoic, or octanoic acid to rescue Tsc2??cell death beneath 48 h of SO strain was determined by flow cytometry. (**) P 0.005; (*) P 0.001. (D) The capacity of 50 mM oleic acid or oleic/linoleic acid to rescue Tsc2??cell death immediately 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 beneath 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.five O2 for 24 h in replete, S, or SG medium had been determined by qRT CR. (G) Tsc2+/+, p53??and Tsc2?? p53??MEFs were grown beneath 21 or 0.five O2 in medium containing 10 mM [U-13C6]glucose or 3 mM [5-13C]glutamine for 24 h, as well as 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 final 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 below normoxic (21 O2) and hypoxic (0.5 O2) situations had been calculated in 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 below S and SO circumstances and also beneath SO conditions were assessed by GC-MS.Young et al.2011). Unlabeled fatty acids in the serum accounted for ;80 of total cellular lipid synthesis in Tsc2?? p53??MEFs under both 21 and 0.five O2, which was surprising in light of reports delineating critical roles for de novo lipid biosynthesis in cancer cell growth (Menendez and Lupu 2007).6-Aminonaphthalene-1,3-disulfonic acid Formula Interestingly, desaturation of de novo synthesized lipids was decreased by 50 in each Tsc2+/+ and Tsc2??cells exposed to 0.Buy941289-27-6 5 O2 as compared with 21 O2 (Fig.PMID:33730845 3H), indicating that lipid desaturation is strongly inhibited by hypoxia and that Tsc2??cells are additional sensitive than Tsc2+/+ cells to reduced levels of desaturated lipids. We confirmed and extended these final results by analyzing the composition of total and de novo synthesized fatty acids under S and SO conditions by gas chromatography-mass spectrometry (GC-MS) (Fig. 3I; Supplemental Fig. S3C). Representative chromatograms of lipid extracts obtained from Tsc2?? p53??MEFs cultured under the many situations are displayed in Supplemental Figure S3C. SO circumstances elicited decrease levels of unsaturated compared with saturated fatty acids (16 and 18 C fatty acids shown), suggesting that availability of unsaturated fat, either from serum or synthesized de novo, is lowered. In addition, the levels of newly synthesized lipids, as indicated by the average enrichment from 13C glucose, were almost equal for 18:0, 18:1, and 18:two fatty acids below S.