Anti nfat1

THP‐1 cells were maintained in RPMI 1640 with 10% FBS (Sigma Aldrich) and peripheral blood monocytes were cultured in X‐VIVO 15 (Lonza) without any supplementation at 37°C in 5% CO₂ atmosphere. The cells were seeded at 1 × 10⁶/ml into the plate (THP‐1) or μ‐Slide VI 0.4 (IBIDI; primary monocytes) and stimulated with ionomycin (1 μg/ml, Sigma Aldrich) for 30–90 min in the presence or absence of CsA (1 μg/ml, Cell Signaling Technologies). THP‐1 cells were then harvested and spun to generate a cytospin preparation, which were fixed by incubation in 4% formaldehyde for 15 min at 4°C. Primary monocytes were fixed in 4% formaldehyde immediately in a μ‐Slide VI 0.4 chamber. BSA (2.5%, Santa Cruz Biotechnology) was used for blocking. Samples were incubated overnight at 4°C with anti‐NFAT1 (#4389, Cell Signaling Technologies) and biotinylated anti‐CD14 (eBiosciences) antibodies that were subsequently detected using AF488 donkey anti‐goat (Thermo Fisher Scientific) and AF647 streptavidin (eBiosciences) secondary antibodies. All antibodies were diluted in DAKO Antibody diluent (DAKO). DAPI (Sigma Aldrich) was used as a nuclear counterstain. Samples were mounted in Mowiol 40–88 (Sigma Aldrich). Images were acquired using a Zeiss LSM 780 confocal microscope with ×40 (1.3 numeric aperture) oil‐immersion objective. Image processing was done using FIJI.33

PBMCs from HD were stimulated for 1 hr with ionomycin at the indicated concentration. They were then stained with a fixable viability dye (ThermoFisher Scientific) and stained for surface markers allowing NK cell identification (CD3, CD19, CD14, and CD56). The samples were then fixed and permeabilised using the Foxp3 Fixation/Permeabilisation concentrate and diluent (eBioscience) and stained with an anti-NFAT1 (Cell Signaling Technology). Sample acquisition was made on an ImageStream X Mark II (Amnis-EMD Millipore, Darmstadt, Germany) with ×40 magnification and analyzed with IDEAS software (v6.0).

PerCP-Cy5.5-conjugated anti-CD4 (RM4-5), anti-CD3ε (145-2C11), and anti-hamster IgG1 (G94-56) were purchased from BD Biosciences (San Diego, CA, USA). APC-conjugated anti-CD69 (H1.2F3) was obtained from eBioscience (San Diego, CA, USA). Alexa488-conjugated anti-CD4 (GK1.5) was obtained from BioLend (San Diego, CA, USA). Anti-pPLCγ1 and anti-NFAT1 were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-PLCγ1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Recombinant Erdr1 was prepared as described previously [38 (link)]. In brief, the bacterial vector with the Erdr1 CDS region was constructed from the Erdr1-pCMV-SPORT6 plasmid (Open Biosystems, Huntsville, AL, USA), and then Erdr1 was purified with over 95% purity from bacteria. Lots with low endotoxin levels (<0.1 EU/mL) determined by the Limulus Amebocyte lysate assay (Cape Cod, East Falmouth, MA, USA) were used for experiments.

ChIP was performed with the ChIP-IT High Sensitivity Kit (Active Motif, 53040). Briefly, a total of 2 × 10⁷ CD4⁺ cells from SRC2^fl/fl or SRC2^fl/fl/CD4^Cre after T_reg differentiation were fixed and sheared as described in the ChIP-IT High Sensitivity manual. ChIP reactions were then performed on 30 μg of the prepared chromatin using specific antibodies (anti-SRC2 from Bethyl or anti-NFAT1 from Cell Signaling Technology) overnight, followed by precipitation with protein G agarose beads. DNA was recovered for sequencing or quantitative reverse transcription PCR to quantify specific DNA fragments that were precipitated. For sequencing, ChIP-enriched samples were sequenced on NovaSeq PE100 at TGen. The analysis was performed through Partek Flow. Briefly, the sequence reads were aligned to the mm10 mouse genome with validation of quality through prealignment and postalignment QA/QC. The enrichment of SRC2 binding sites across the genome was analyzed using MACS2. The primers used for RT-qPCR are listed in table S1.

MiaPaCa-2 and BxPC-3 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). L3.6pl cells were obtained from MD Anderson Cancer Center. All cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a 5% CO₂ atmosphere at 37 °C. The cell lines have been tested and authenticated in a core facility of the Applied Genomics Technology Center at Wayne State University. The method used for testing was short tandem repeat (STR) profiling using the PowerPlex^® 16 System from Promega (Madison, WI. USA). RP4010 (Rhizen Pharmaceuticals S.A., La Chaux-de-Fonds, Switzerland) was dissolved in DMSO to make a 50-mM stock solution; 1-mM stock solutions of gemcitabine (Gemzar, Eli Lily, Indianapolis, IN, USA) and nab-paclitaxel (Abraxane, Celgene, Summit, NJ, USA) were also made in DMSO. Anti-NFAT1 (Cell Signaling, Danvers, MA, USA), anti-p-Akt (Cell Signaling), anti-p-4EBP1 (Cell Signaling), anti-S6K (Cell Signaling), anti-p-S6K (Cell Signaling), anti-NF-κB (MilliporeSigma, Burlington, MA, USA), anti-β-actin (Cell Signaling), and anti-GAPDH (Thermo Fisher Scientific, Waltham, MA, USA) primary antibodies were used for Western Blot analysis.