Fig. 6: Glucosamine supplementation rescues αβ-T cell development in fetal thymic organ cultures.
WT or GFAT1T−/− mice were used for the following experiments. Thymic lobe from fetus was incubated in complete media containing glucosamine (GlcN) with or without dimethyl-2-ketoglutarate (DKG) and the adjoining lobe was cultured in complete media only as control. Thymocytes were stained for CD4, CD8, CD25, CD44, TCRβ, and γδTCR and analyzed by flow cytometry. a Plot and bar graph represent proportion of subsets expressing either surface TCRβ (αβ-lineage) or γδTCR (γδ-lineage) after 7 days of culture (n = 3–5 fetal lobes). Plot is representative of three experiments with similar results (a). b Bar graph showing TCRβ and γδTCR levels (median fluorescence intensity; MFI) on the surface of αβ- and γδ-lineage-committed thymocytes, respectively (n = 3–5 fetal lobes) and representative FACS plots of the respective receptor staining from three experiments with similar results are shown. c Bar graphs (median fluorescence intensity; MFI) (n = 3–5 fetal lobes) and FACS plots of TCRβ levels expressed on the surface of each subset (c) The inset is a blowup of TCRβ expression on DN to DP stages. FACS plots are representative of three experiments with similar results. d Thymocyte subsets expressing either CD4 or CD8 were plotted. FACS plots are representative of three experiments with similar results. Bar graphs show proportion of different thymocyte subsets (n = 3–5 fetal lobes). e Immature CD4−CD8−Lin− DN thymocytes were stained for CD25 and CD44 expression. Representative FACS plots out of three experiments with similar results showing relative number of DN subsets are indicated in each quadrant and plotted in the bar graph (n = 3 fetal lobes). All graphs from (a–e) are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by Tukey’s (a, b) or Šidák’s (c–e) post-hoc test. See also Supplementary Fig. 6. Source data are available for (a–e).
