Fig. 1: NAT10 is upregulated during T cell activation.

a, NAT10 expression in CD3⁺ T_N cells stimulated with anti-CD3/CD28 for the indicated lengths of time. b, Dot blot and densitometry analysis of ac⁴C modifications in total RNA (500 ng) of CD3⁺ T_N cells stimulated with anti-CD3/CD28 for the indicated lengths of time. Left, representative anti-ac⁴C dot blot. Right, bar graph showing the relative ac⁴C intensities normalized to total RNA quantified by methylene blue; n = 3; *P = 0.0153, **P = 0.0039 and ***P = 0.0007. c, Schematic of the acute LCMV infection model in WT mice. Eight-week-old WT mice were intraperitoneally injected with 2 × 10⁵ p.f.u. LCMV (Armstrong strain) and killed at 2, 4, 6, 8, 12 and 16 days after infection. Mice treated with equal volumes of PBS served as controls. Serum RNA and CD3⁺ T cells were isolated for further analysis; D, day; i.p., intraperitoneal. d, Changes in serum viral load during infection; n = 3. e, Western blot and densitometry analysis of NAT10 protein expression in T cells of LCMV Armstrong-infected mice at the indicated times. Each blot represents an independent biological sample. f, Schematic diagram of TCR, IL-2 and IL-7 signaling pathways during T cell activation. Image created using BioRender. g, NAT10 and c-JUN expression in CD3⁺ T_N cells stimulated with TCR (anti-CD3/CD28), IL-2 and IL-7 for the indicated lengths of time. h, IGV snapshots showing c-JUN, JUND, JUNB, NFAT2, P65 and FOSL2 binding sites at the Nat10 locus. The arrow indicates the start site and transcription direction. i, NAT10 expression in CD3⁺ T_N cells stimulated with anti-CD3/CD28 for 24 h in the presence of NFAT inhibitor (NFATi), c-JUN inhibitor (c-JUNi), P65 inhibitor (P65i) and an equal volume of DMSO at the indicated concentrations (in μM), respectively. j, Nat10 expression at the transcriptional level under the same conditions as in i; n = 3. The following are the exact P values from left to right: ***P = 0.0004, ***P = 0.0007, ***P = 0.0004 and not significant (NS), P > 0.05; Con, control. k, c-JUN ChIP–qPCR for the Nat10 locus in CD3⁺ T_N cells stimulated with anti-CD3/CD28 for 24 h; n = 3; ****P < 0.0001. n refers to the number of biologically independent samples. Error bars represent mean ± s.e.m. (b, d, e, j and k). Representative data of three independent experiments are presented (a, b, d, e, g and i). Data were analyzed by two-tailed, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (b and j) and two-tailed, unpaired t-test (k).

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