Extended Data Fig. 4. Further characterization of stem cell and embryo metabolic profiles.
(a) Energetics of normal and mTORi-treated ESCs and TSCs. Seahorse glycolysis (ECAR) and basal respiration (OCR) assays were performed on normal or mTORi-treated cells (4 days). Three technical replicates of two biological replicates are shown. Fatty acid oxidation rate in normal and mTORi-treated (48 hours) ESCs and TSCs with or without the CPT1 inhibitor. FAO Blue reagent was used for measurement of FAO output on live cells. The area-corrected median cytoplasmic FAO Blue levels for two biological replicates are shown. (b) Energy preference of paused ESCs. Seahorse Mito Fuel Flex test was performed on normal and paused ESCs (4 days). Data from two biological replicates are shown. Average of two replicates are indicated with the bars. (c) Survival curves or paused embryos supplemented with vitamin C, alpha-ketoglutarate, free short chain fatty acids, and short chain carnitine-conjugated fatty acids. n = number of embryos. Statistical test is the G-rho family test of Harrington and Fleming89. (D-H) IF staining against phospho-mTOR/OCT4 (d), pS6/OCT4 (e), pAKT/OCT4 (f), pAMPK/OCT4 (g), and pACC/OCT4 (h) in E4.5, D5 mTORi-only, mTORi+c, and mTORi+CPT1i embryos. Scale bar: 50 µm. Violin plots show the respective quantifications of the area-corrected cellular signal intensity. n = number of cells. a and b: p < 0.05 compared to E4.5 and D5 mTORi, respectively, computed with a one way ANOVA and Tukey HSD posthoc test. Minimum 4 embryos were used.