Extended Data Fig. 1: Characterization of NFAT5mCherry reporter mice.
a) Fold change of Nfat5 expression level in Tpex or Tex over indicated populations (left). Mean Nfat5 expression level of various CD8+ T cell populations in indicated studies assessed by TILatlas (right). b) NFAT5 reporter mouse strain: The TAG stop codon in exon 14 of the mouse Nfat5 gene was replaced by CRISPR/Cas-mediated genome engineering with the P2A-mCherry cassette to create a knock-in NFAT5-P2A-mCherry reporter model in C57BL/6 mice. c) Example of a gating strategy used throughout the study. d–f) Spleen (d), lymph node (e), and thymus (f) from WT, NFAT5m/wt and NFAT5m/m mice were collected and analyzed for their immune compartment, and thymic development. (e) by flow cytometry. Each dot represents one mouse. g, Relative expression (2-deltaCq) of mCherry and Nfat5 assessed by RT-PCR and normalized to β-2-microglobulin of NFAT5m/wt or NFAT5m/m CD8+ T cells cultured for 72 h under different hyperosmolar conditions ranging from 300 mOsm/kg to 500 mOsm/kg. Each dot represents the mean from a technical replicate from cells from one mouse. h) Histogram showing mCherry expression of NFAT5m/m CD8+ T cells from the lymph node in comparison to a littermate control mouse (WT). i) Western blot showing the total protein level of NFAT5flox/flox CD4-Cre−/− (WT), CD4-Cre+/− NFAT5flox/flox (NFAT5KO), NFAT5m/wt and NFAT5m/m P14 CD8+ T cells cultured in complete RPMI with 1 uM GP33 peptide and 20 U/ml rhIL-2 at 420 mOsm/kg- for 72 hours. β-actin serves as a housekeeping gene. Ratio of NFAT5 level on β-actin has been calculated after quantification of the bands with ImageJ. Biological duplicates shown for each condition. This experieent is representative of 3 independent experiments.
