*3.10. Immunofluorescence*

Splenocytes were seeded in a 24-well plate at a density of 5 × 10<sup>6</sup> cells/well, incubated with various concentrations of Compound **2** or CsA, and then activated by culturing with Con A (5 μg/mL) for 48 h. After treatment, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized in 0.5% Triton X-100 for 15 min, blocked with 5% BSA for 1 h, and then incubated overnight in the respective primary antibody; NFAT1 (D43B1) XP® Rabbit antibody was used at a 1:100 dilution in PBS. The corresponding fluorescent anti-rabbit IgG (H+L) secondary antibody was added for 1 h. Nuclear staining was performed with the addition of 300 μL DAPI for 5 min. Stained cells were washed with PBS and visualized using confocal microscopy.

#### *3.11. Real-Time Quantitative PCR*

Total cellular RNA was isolated from splenocytes (5 × 10<sup>6</sup> cells/well) treated with the indicated concentrations of compounds or cyclosporin A for 48 h and extracted using RNA isolate. The obtained total RNA (2 μg) was used for cDNA synthesis with a Servicebio®RT First Strand cDNA Synthesis Kit with random primers, according to the manufacturer's instructions. qPCR was performed ith SYBR Green qPCR Master Mix (High ROX) using ABI StepOne Plus Real-time Detection System and *β*-actin as internal control. The primers were as follows: IL-2 forward, 5--TGTCACAAACAGTGCACCTACTTC-3-; IL-2 reverse, 5--TGTGGCCTTCTTGGGCATGT-3-; *β*-actin forward, 5--GTGACAGCAGTCGGTTGGAG-

3- ; *β*-actin reverse, 5--AGTGGGGTGGCTTTTAGGAT-3-. The expression levels of genes were normalized to the expression of *β*-actin mRNA and analyzed using the delta-delta CT method (2−ΔΔCT).

## *3.12. IL-2 ELISA Assay*

Splenocytes were treated as described in the RT–qPCR section, and then the IL-2 levels in the obtained culture supernatants were measured using an enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's instructions. Cytokine standard curves were used to calculate the amount of IL-2, and the absorbance of each well was read at OD450 using an ELISA reader.

## *3.13. Molecular Docking Analysis*

Molecular docking studies were performed to investigate the binding mode of Compound **2** with calcineurin using AutoDock 4.2 software. The crystallographic structure of calcineurin (PDB ID: 1AUI) was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB, http://www.rcsb.org, accessed on 17 December 2021). The structure file 1AUI was protonated, and water was deleted at pH 7 using the Clean Protein tool. The 3D structure of the small molecule was built using ChemBioDraw Ultra 14.0 (Cambridgesoft Corp., Cambridge, MA, USA) and optimized using MM2 and the steepest gradient method in Chem3D Ultra 14.0 (Cambridgesoft Corp., Cambridge, MA, USA). The Lamarckian genetic algorithm method was used in AutoDock 4.2 (Scripps Research, San Diego, CA, USA), and a docking site was defined as all residues within an RMS tolerance of 1.0 Å. The default parameters were used if no other parameters are mentioned. The obtained results were analyzed and visualized with PyMOL software (Schrödinger, New York, USA).
