Fig. 2: Gln and Arg are the main carbon sources for GAB biosynthesis in Teff cells.
From: GAB functions as a bioenergetic and signalling gatekeeper to control T cell inflammation
a,b, ABAT protein levels were determined by IB (ABAT molecular weight, 56 kDa; full-scan images are provided as source data) (a) and cytometry (b). c, As illustrated by the experimental scheme (left), the indicated metabolites in TH17 cells were quantified by GC–MS (right); data are representative of n = 3 biologically independent samples. d, Scheme of the GAB metabolic pathways and a pharmacological inhibitor Vig of ABAT. e, Schematic diagram of GAB biosynthesis from Arg (left), with the expression of relevant genes determined by qPCR detection (right). mRNA levels for Tnai cells were set to 1. The heatmap represents the relative mRNA expression level (see colour scale). Values and s.d. are provided as source data. f. As illustrated by the experimental scheme (left), the indicated metabolites in TH17 cells were quantified by YSI (Gln and Glu) or by bioassay kits (Arg and GAB) (n = 3 biologically independent samples). g, Diagram of conversion of [13C5]Glu and [13C6]Arg to downstream metabolites (top). Indicated metabolites in TH17 cells were quantified by GC–MS (n = 3 biologically independent samples) (bottom). Numbers along the x axis represent those of 13C atoms in the given metabolites. h, Diagram of the conversion of [13C6]Glc to downstream metabolites. Indicated metabolites in TH17 cells were quantified by GC–MS (n = 3 biologically independent samples). Black dot, 12C; red dot, 13C derived from [13C6]Glc. i, Diagram of the conversion of [13C4]Put to GAB. Indicated metabolites in TH17 cells were quantified by GC–MS (n = 3 biologically independent samples). Unpaired two-tailed Student’s t-test. j, As illustrated by the experimental scheme (top), cytokine expression and cell viability were determined by flow cytometry (bottom). All experiments with bicuculline (5 μM); complete medium, 2 mM Gln + 0.1 mM Arg; Gln (low), 10 μM Gln + 0.1 mM Arg; Gln (low plus GABA), 10 μM Gln + 0.1 mM Arg + 1 mM GABA; Arg (low), 2 mM Gln + 10 μM Arg; Arg (low plus GABA) 2 mM Gln + 10 μM Arg + 1 mM GABA. k, Statistical analysis for j; data are representative of n = 3 biologically independent samples. Significance was calculated by one-way ANOVA with Tukey’s multiple-comparisons test. Gln, glutamine; Glu, glutamate; Arg, arginine; ODC, ornithine decarboxylase; DAO, diamine oxidase; PDH, pyrroline dehydrogenase; M, million.
