Fig. 3: Effect of ARE-interlinked m⁶A sites on mRNA stability.

A Actinomycin D experiments showing % of remaining mRNA in CD8⁺ T cells treated with DMSO or METTL3inhibitor. Mean ± SD; n = 3 (one donor provided material for 0,2 h). Mixed-effects model (REML), p-values indicate treatment effects. Donors were processed in parallel. Representative results from three independent experiments (IL7R, TNF). B mRNA levels IL7R (n = 5), CCL4 (n = 5), CD69 (n = 4), CD28 (n = 4), TCF7 (n = 3), ZFP36L1 (n = 3), TNF (n = 4), IFNG (n = 4) in CD8⁺ T cells treated with METTL3inhibitor. Fold change relative to DMSO (grey line). Reference gene 18S. Mean ± SD; paired two-tailed t tests on ΔCt values. For TNF, one Grubbs-identified outlier is displayed but excluded from statistics. C, D Representative flow cytometry plots illustrating IL7Rα gating (C) and quantification of IL7Rα⁺CD8⁺ T cells (D) (n = 10). Paired two-tailed t tests. E, F Representative histogram (E) and quantification (F) of IL7Rα median fluorescence intensity (MFI) in IL7Rα⁺ cells treated with METTL3inhibitor relative to DMSO (grey line) (n = 10). Mean ± SD; one-sample two-tailed t test. G GFP MFI in T cells transduced with GFP-3’UTR constructs (S3H) after treatment with FTO inhibitor followed by PMA/ionomycin activation relative to DMSO (n = 3). One-way ordinary ANOVA. H, I IFNγ (H) and TNFα (I) protein levels measured by ELISA in DMSO (0 μM) or FTOinhibitor treated cells (n = 3). RM one-way ANOVA. J, K Representative amplification curves (ΔRn) and Ct quantification for reference (J) and miCLIP-ARE (K) amplicons in control (grey) and METTL3-KO (pink) samples (n = 3). Mean ± SD. L Fold-change expression (ΔΔCt) for the miCLIP-ARE site in METTL3-KO and FTO-KO cells relative to control (grey line). Mean ± SEM; unpaired two-tailed t test comparing METTL3-KO and FTO-KO; paired t test for control vs METTL3-KO on ΔCt values; (n = 3). M–P mRNA levels (M) and GFP protein levels (N–P) in CD8⁺ T cells transduced with GFP-3′UTR constructs (S3O). mRNA levels normalised to 3′UTR-TNF_WT (grey line) (n = 10); one-way ANOVA with Holm–Šídák correction (M). Representative plots of resting or PMA/ionomycin-activated cells (N). GFP MFI quantification in activated cells (O, P) (n = 15); paired two-tailed t tests. Box-and-whisker plots (min–max, median, 25th–75th percentiles). All n values reflect distinct donors. Source data are provided as a Source Data file.