Fig. 7: Dietary supplementation of GFAT1^T−/− mice with glucosamine and α-ketoglutarate partially restores αβ-thymocyte development by increasing the viability of DN and SP cells.

Three-week-old male and female GFAT1^T+/+ (WT) or GFAT1^T−/− littermates were fed with regular water or water containing GlcN and DKG (100 mM each) (GlcN/DKG-supplemented GFAT1^T−/− mice are also referred to as GD+) and thymocytes were harvested after 1 month. Cells were stained for Lin, CD4, CD8, CD25, CD44, Annexin V, TCRβ and γδTCR and analyzed by flow cytometry (a–f, h–k) or subjected to metabolite analysis (g). a Total cell number for each mice are indicated (n = 3 mice each). b, c Proportion of subsets expressing either high TCRβ or high γδTCR on their surface are shown in representative plots from three experiments with similar results and bar graph (b). Absolute numbers of αβ-lineage cells are shown (c). (b, c, n = 3 mice with 3 technical replicates each). See also Supplementary Fig. 7a. d, e Thymocyte subsets expressing either CD4 or CD8 were plotted. FACS plots are representative of three independent experiments with similar results. Bar graphs represent the proportion (d) and absolute number (e) of each subset (d, e, n = 3 mice each with 3 technical replicates each). Insets are blowups of the absolute number of CD8-ISP and CD8-SP cells. f Bar graph (median fluorescence intensity; MFI) of TCRβ expressed on the surface of each thymocyte subset with corresponding FACS plots. Inset is a blowup of TCRβ expression on DN to DP stages (n = 3 mice each with 3 technical replicates each). Representative FACS plots are from three experiments with similar results. g Metabolites were isolated from thymocytes and analyzed by LC/MS. Bar graphs represent fold changes of indicated metabolite relative to WT (n = 4 independent samples each). See also Supplementary Fig. 7c. h, k Immature CD4⁻CD8⁻Lin⁻ DN thymocytes were stained for CD25 and CD44 expression. Relative subset numbers are indicated in each quadrant and plotted in the bar graph (h). Representative FACS plots are shown from three experiments with similar results. Total cell numbers from each DN subset were plotted (i). The viability of TCRβ^–DN3 and TCRβ⁺-DN4 was plotted (j). Bar graph of TCRβ expression (median fluorescence intensity; MFI) on the surface of DN4 thymocyte and representative FACS plot from three experiments with similar results are shown (k). n = 3 mice each with 3 technical replicates each (h–k). All graphs from (a–k) denote mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by Šidák’s (a–f, g, j, k) or Tukey’s (h, i) post-hoc test. Source data are available for (a–k).