Extended Data Fig. 6: Effect of mTORC1 and Gln on Asn-mediated CD8+ T cell activation.
From: Asparagine enhances LCK signalling to potentiate CD8+ T-cell activation and anti-tumour responses
a, Phosphorylation of the mTORC1 effector S6 (phospho-S6 Ser 235/236) in mouse CD8+ T cells left unstimulated (naive) or activated in the presence of increasing amount of Asn. b, Western blot analysis of mouse naive CD8+ T cells left untreated (naive), or activated in Ctrl (Asn/Asp-free) medium or Asn medium supplemented with increasing amounts of rapamycin for 12 h. c, Surface expression of CD69, CD25, and CD44 on mouse naive CD8+ T cells stimulated in Asn/Asp-free medium (Ctrl) or Asn medium with 10% dialysed FBS and increasing amounts of rapamycin (n = 3 independent wells). d, Mouse naive CD8+ T cells stimulated in Asn/Asp-free (-) or Asn-supplied (Asn) medium without (0) or with increasing amounts of Gln for 24 h were treated with 40 μg/ml Puromycin for another 10 min. Medium was added with 10% dialysed FBS. Puromycin incorporation was measured by FACS analysis using FITC-conjugated anti-Puromycin antibody (n = 3 independent wells). Representative flow cytometry plots are shown. e, Mouse naive CD8+ T cells were stimulated in the indicated medium with 10% dialysed FBS for 24 h. MG132 (10 μM) was added 2 h before harvesting for western blot analysis. f, Surface expression of CD69, CD25, and CD44 on mouse naive CD8+ T cells stimulated in Asn/Asp-free (-) or Asn medium containing no (0) or increasing amounts of Gln for 24 h (n = 3 independent wells). All medium was added with 10% dialysed FBS. All data are mean ± SEM, two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001; ns, not significant. Data in a, b and e are representative of three independent experiments. Related representative FACS plots (Supplementary Figs. 16, 17), uncropped blots for a, b, e, and numerical source data for c, d, f, are provided.
