Fig. 3: Extracellular Met availability regulates Ca²⁺-mediated NFAT1 activity.

a, Representative plot of Indo-1 analysis of Ca²⁺ flux of either CD8⁺ T cells naive or activated with anti-CD3 and anti-CD28 by anti-hamster IgG crosslinking in either 0.1 mM or 0.03 mM Met-containing Ca²⁺-free Ringer solution with addition of 2 mM Ca²⁺ to measure Ca²⁺ influx (n = 3 biological replicates per group). iono, ionomycin. b,c, Area under the curve (AUC) (b) and maximum peak signal (c) of the calcium flux from a, normalized to values of activated cells in 0.1 mM Met (n = 3 biological replicates per group). d, Representative images of T cells, cultured with or without anti-CD3/28 Dynabeads (dark gray masked), in 0.1 mM and 0.03 mM Met, with or without CsA treatment for 30 min, stained for NFAT1 (red), Hoechst (blue) and Phalloidin (green) (dashed lines indicate nuclei). The identical figure without masking or nuclear demarcation is shown in Extended Data Fig. 5a. e, Quantification of NFAT1 intensity as nuclear to total cell ratio (c). Each circle represents one cell, n = 40 cells per group. Scale bar, 10 μm. f, Histogram of NFAT1-binding signals (read count per million reads normalized to background) from NFAT1 CUT&RUN on T cells initially activated in 0.1 or 0.03 mM Met for 30 min and assessed 24 h postactivation. g,h, NFAT1 CUT&RUN peaks at the known target genes (g) and quantification of known NFAT1-binding regions of Lag3, Pdcd1, Havcr2, Ctla4 and Tnfrsf9 and Tox (h, normalized read count (see differential binding analysis)) in T cells initially activated in 0.1 or 0.03 mM Met for 30 min and assessed 24 h postactivation (n = 2 biological replicates per group). Data are mean ± s.d. Boxplots shows minimum and maximum value with median as center. Paired two-tailed Student’s t-test (b and c), unpaired two-tailed Student’s t-test (e).

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