Fig. 2: Defective expression of key surface receptors at the DN stage in GFAT1-deficient thymocytes.
Thymocytes from male and female WT (Lck-Cre−/−/Gfat1f/f) and knockout (Lck-Cre+/−/Gfat1f/f; referred to as GFAT1T−/−) littermates were stained for Lin, CD4, CD8α, CD25, CD44, CD27, Notch1, and TCRβ followed by flow cytometric analysis to measure the proportion of Lin− DN subsets as indicated in each quadrant (a-f, i) or further stained for intracellular (ic) TCRβ (g-h) before flow cytometry. a, b FACS plots and bar graphs represent proportion (a) or absolute cell number (b), (n = 9 mice). c, d DN3 thymocytes were further analyzed for the proportion of CD27−DN3a and CD27+DN3b subsets (see also Supplementary Fig. 2a) followed by analysis of CD27 and Notch1 surface expression. Bar graphs of median fluorescence intensity (MFI) of CD27 (c) and Notch1 (d) expressed on the surface of DN2, DN3a, DN3b, and DN4 thymocytes (n = 3 mice with 3 technical repeats) and respective representative FACS plots are shown. e–g Representative FACS plots showing DN3b and DN4 subsets that express Notch1 and TCRβ is shown (e). Percentage is indicated in each quadrant. Representative FACS plots and bar graphs represent median fluorescence intensity (MFI) of TCRβ expression on the surface of DN3b and DN4 cells (f)(n = 9 mice) or ic-TCRβ expression (g)(n = 6 mice). h DN3b and DN4 subsets were analyzed for ic- and surface (s) TCRβ staining. Representative FACS plots with the percentage of cells in each quadrant are shown. i CD25 expression on the surface of DN2, DN3a and DN3b thymocyte subsets. Bar graph of median fluorescence intensity (MFI) and respective representative FACS plots of CD25 staining are shown (n = 9 mice). FACS plots from (a–i) are representative of at least 3 experiments with similar results. All data (a–d, f, g, i) are mean ± .SD. *p < 0.05, **p < 0.01, ***p < 0.001 using a two-sided Student’s t test. Source data are available for (a–i).
