Fig. 3: Defects in hexosamine, glycosylation, and increased unfolded protein response in GFAT1-deficient thymocytes.

Thymocytes from male and female age-matched WT, GFAT1^T−/− and /or rictor^T−/− mice were used for the following except for b. a Metabolites were extracted and analyzed from equivalent thymocyte numbers. Graphs represent mean fold changes relative to WT (n = 5 independent samples; [1 WT, pool of 3-5 GFAT1^T−/− and 2–4 rictor^T−/− per sample). Error bars denote SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by Tukey’s post-hoc test. See also Supplementary Fig. 3b. b Thymocytes were harvested from male and female OT-1/WT and OT-1/GFAT1^T−/− mice and treated with vehicle or MG132 for 4 h. Lysates were subjected to lectin pull-down assays followed by immunoblotting of TCRβ. Fold changes relative to TCRβ expression in untreated OT-1/WT cells (lane 1 for input, lane 5 for pull down) are indicated below each blot. Representative blots from two independent experiments with similar results are shown. c–e Thymocytes were harvested, intracellularly stained (c) or surface stained (d, e), then analyzed by flow cytometry. Bar graphs represent mean fluorescence intensity (MFI). n = 5–8 mice for (c–e) and the respective representative FACS plots from three experiments with similar results are shown. All data 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. f, h Protein extracts of thymocytes were resolved and subjected to immunoblotting. β-actin was used as loading control for all blots. Shown are representative immunoblots from at least three independent experiments with similar results. Closest MW (KD) marker is indicated for each blot. g Protein extracts from thymocytes were subjected to quantitative proteomics and data were analyzed by Ingenuity Pathway Analysis (IPA). Shown is the heat map of statistically significant (p < 0.05 as determined using two-sided Student’s t test) canonical metabolic pathway alterations in GFAT1- and rictor-deficient thymocytes relative to WT. Red asterisks denote an increase of both tRNA charging and salvage pathways of pyrimidine. Pathways are ranked according to the z-score that predicts upregulation (orange) or downregulation (blue). See complete heat map in Supplementary Fig. 3d. Source data are available for a-f,h.