Extended Data Fig. 6: Initial Met limitation alters KCa3.1 methylation, but not DNA or histone methylation.

a, Methylation potential of 10 ng/ml SIINFEKL-activated OT-I T cells in 0.1 mM or 0.0 mM Met at 10, 30 and 60 min. b, %5-methylcytosine (%5-mc) of OT-I CD8⁺ T cells activated with 2.5 ng/ml SIINFEKL in 0.1 mM or 0.03 mM Met for 30 min, then 0.1 mM Met for 24 h. c, d, Histograms (top) and heat-map (bottom) of H3K4me3 (c) and H3K27me3 (d) CUT&RUN (read count per million, normalized to background) in OT-I CD8⁺ T cells activated as in (b) at 24 h post activation. e, Unedited image of Fig. 4a with unmasked, red CD3/28 Dynabeads (grey in main Figure to highlight me-Arg staining). f, Conserved arginine (red) in KCa3.1, analyzed by protein-BLAST and visualized using Jalview. g, Tetrameric assembly of human KCa3.1-Calmodulin complex in membrane (left). KCa3.1 is represented as ribbons, calmodulin quartet as cartoon, interacting Ca²⁺ ions as purple spheres, and K⁺ ions as cyan spheres. KCa3.1 monomer (blue) shown in complex with calmodulin (yellow) and demethylated arginine-352 (green) (right). h, MD simulations analysis. Illustration of total counts of negatively charged residues within a 4 Å radius of all three R352/SDMA/ADMA of huKCa3.1 over simulation time (averaged across all four monomers, three MD trials). i, Representative plot of Fluo-8 AM analysis of Ca²⁺ flux in CD8⁺ T cells activated by IgG crosslinking anti-CD3 and anti-CD28 in Ca²⁺-free Ringer solution with either 0.1 mM or 0.03 mM Met and treated with either DMSO or 1 μM TRAM-34 (n = 2 mice/group). j, NFAT1 quantification (nuclear to total cell ratio) in OT-I CD8⁺ T cells activated for 30 min with anti CD3/28 Dynabeads in 0.03 mM Met (left) or 0.1 mM Met (right) in 0.5, 1 or 2 μM TRAM-34 (each circle=one cell, n = 27–45 cells/group). k, Schematic design for OT-I T cell activation in 0.1 or 0.03 mM Met, treated with DMSO or TRAM-34 for 30 min, followed by washing and culturing in 0.1 mM Met plus SIINFEKL (2.5 ng/ml) for 24 h before injection into B16-OVA tumour-bearing Rag1^−/− mice. (a–d: n = 3 mice/group) Data are mean±s.d. Unpaired one-tailed Student’s t-test (a) or unpaired two-tailed Student’s t-test (b, j). Illustration in k created with Biorender.com.

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