Fig. 1: Defective αβ-T cell development in the absence of GFAT1. | Nature Communications

Fig. 1: Defective αβ-T cell development in the absence of GFAT1.

From: Dietary glucosamine overcomes the defects in αβ-T cell ontogeny caused by the loss of de novo hexosamine biosynthesis

Fig. 1

a Total cell extracts from thymocytes, lung, and liver from male and female WT (+/+) or mice with specific deletion of GFAT1 in T cells (GFAT1T−/−) were subjected to SDS-PAGE and immunoblotted using the indicated antibodies. Representative blot out of two independent experiments with similar results is shown. b Thymocytes from male and female 5-week-old Lck-Cre−/−/Gfat1f/f (WT), Gfat1+/+/Lck-Cre+/− (Lck-Cre+/−) or Lck-Cre+/−/Gfat1f/f (GFAT1T−/−) littermates or age-matched were harvested and counted by trypan blue exclusion. Each symbol represents one mouse (n = 4–8 mice each). c–f Splenocytes of 5-week-old male and female Lck-Cre−/−/Gfat1f/f (WT), Gfat1+/+/Lck-Cre+/− (Lck-Cre+/−) or Lck-Cre+/−/Gfat1f/f (GFAT1T−/−) littermates or age-matched were stained for TCRβ, CD4 and CD8α followed by flow cytometric analysis. Shown are the proportions of T cells among splenocytes (c); total number of T cells in each mouse (d) (n = 4–6 mice each); proportions of CD4 T helper (CD4+ TH) and CD8 T cytotoxic (CD8+ TC) cells among peripheral T cells (e); total cell number of CD4+ TH and CD8+ TC subsets in the spleen of the respective mice (f) (n = 4–6 mice). Representative FACS plots (c, e) from three experiments with similar results are shown. g–j Thymocytes from male and female Lck-Cre−/−/Gfat1f/f (WT), Lck-Cre+/− (Lck-Cre+/−) or Lck-Cre+/−/Gfat1f/f (GFAT1T−/−) littermates or age-matched were stained for CD4, CD8α, CD147 & TCRβ followed by flow cytometric analysis to measure the proportion of thymocyte subsets as indicated in each quadrant. Bar graph represents proportion (g) or absolute cell number (h) (n = 4–8 mice). CD8+CD4− thymocytes were gated for TCRβlow/CD147+ to distinguish CD8-ISP (TCRβlow) from lineage-committed CD8-SP (TCRβhigh) cells (i). Bar graph (j) represents absolute cell numbers of CD8-ISP and CD8-SP (n = 4–8 mice). Representative FACS plots (g, i) from three experiments with similar results are shown. All data from (b–d, f–j) are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by Tukey’s post-hoc test. Source data are available for (a–j).

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