Extended Data Fig. 4: XBP1 controls the abundance of glutamine transporters in glucose-deprived T cells.

a, b, Pre-activated wild-type or XBP1-deficient CD4⁺ T cells were incubated in the indicated medium for 6 h and then stained on poly-l-lysine coated discs using antibodies specific for ASCT2 (green) or SNAT2 (red). Nuclei are depicted in blue (DAPI staining). a, Representative confocal images of the indicated T cells from three experiments. b, The mean fluorescence intensity (MFI) of each glutamine transporter on about 50 individual cells from three independent slides (n = 150) was computationally quantified using the ImageJ software by two independent investigators in a blinded manner. Individual dots depict the average MFI of each independent analysis (n = 6). c, Naive splenic CD4⁺ T cells isolated from wild-type or XBP1-deficient mice were activated by CD3 and CD28 stimulation for 48 h and then incubated in medium that lacks glucose for 6 h. mRNA expression of genes encoding glutamine transporters was determined by qPCR (n = 6 from three experiments). Data were normalized to endogenous expression of Actb in each case. d, e, Pre-activated mouse CD4⁺ T cells were incubated in the indicated medium for 6 h in the presence or absence of proteasome inhibitor MG132 (10 μM). d, Protein levels of the glutamine transporter SNAT1 were determined by immunoblot analysis, in which β-actin was used as loading control. Representative image from five independent experiments. e, Densitometric quantification of SNAT1 (n = 5). Results are presented as relative expression compared with untreated control T cells incubated in glucose-containing medium. Data are shown as mean ± s.e.m. (b, c). n values represent biologically independent samples (c, e). Two-tailed Student’s t-tests (b); two-tailed paired Student’s t-tests (e); *P < 0.05, ***P < 0.001; NS, not significant.

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