Fig. 2: Asparagine deprivation tailors differentiation and metabolic adaptation in a ROS-dependent manner.
a, The enriched transcription factors (TFs) in both Asn-free and control conditions are selected and the TFs that have the most target region enrichment in either Asn-free or control are represented as the preference. b,c, Naive CD8+T cells were cultured under CD8 differentiation conditions in both control medium and asparagine-deprived medium for various durations. Subsequently, CD8+T cells were stained for DCFDA (b) and MitoSOX (c) (n = 3). d, Lysed cells from a control medium or an asparagine-deprived medium 3-day cultured CD8+ T cell. The cells were subsequently analysed using liquid chromatography–mass spectrometry to assess the GSSG/GSH ratio (n = 3). e, Naive CD8+T cells were cultured under CD8+ differentiation conditions in both control medium and asparagine-deprived medium for various durations. The protein levels of the indicated molecules were determined through immunoblot analysis, which was independently repeated three times. Blots were cropped for clarity. Actin was run on separate gels due to different protein loading amounts. To ensure comparability, all blots were processed in parallel under identical conditions. IB, immunoblot. f, Naive CD8+T cells cultured in CD8+ differentiation conditions with either asparagine-free or control medium and added with 20 mM NAC for 3 days, CD8+ T cells stained for IFNγ and granzyme B and analysed by flow cytometry (n = 3). PE, phycoerythrin; APC, allophycocyanin. g, Imaging of NFAT localization staining (in green) and DAPI (in blue) in CD8+ T cells by confocal microscopy (n = 3). Scale bar, 5 µm. All data are mean ± s.e.m. and were analysed by one-tailed unpaired (d) Student’s t-test, one-way analysis of variance (ANOVA) with two-way ANOVA with Tukey’s multiple comparisons test (f and g) or Sidak multiple comparisons tests with adjusted P value (b and c), and data are cumulative results from three independent experiments. NES, normalized enrichment score.
