Stattic sc 202818

STATTIC (sc-202818, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SP600125 (s5567) and Anisomycin (A9789) purchased from Sigma (Louis, MO, USA) were dissolved in 100% dimethyl sulfoxide to prepare a 40 mM stock, 20 mM stock and 15 mg/ml stock respectively, and stored at -20°C. Recombinant human interleukin (IL)-6 purchased from R & D Systems (206IL, Minneapolis, MN, USA) was reconstituted in sterile PBS containing 0.1% bovine serum albumen to prepare a 10 μg/ml stock and stored at -20°C. The stock solution was added in culture medium to achieve the indicated final concentrations for cell culture.
The cell proliferation was examined using a CCK-8 cell proliferation kit (Dojindo Laboratories, Kumamoto, Japan), according to the instruction from the supplier. Absorbance was measured at 450 nm with Microplate Reader (Bio-Rad, La Jolla, CA, USA). After 24 hr serum depletion (0.2% FBS), cells were subsequently incubated in 10% FBS medium for an additional 24 hr before harvest. Cell cycle assessment and data analysis were carried out referring to our previous report
[6 (link)] using FACS Calibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with the ModiFit LT v2.0 software.

The ADORA2A-knockout (gKO) mice used in the study were obtained from Dr. Jiang-Fan Chen (Molecular Neuropharmacology Laboratory, Boston University School of Medicine, Boston, MA, USA). Wild-type (WT) C57 mice were purchased from the Department of Laboratory Animal Science, Army Medical University, China. All mice in this study were 8–9 weeks old and weighed 25–28 g. All animal experiments and care were approved by the Third Military Medical University Committee on Ethics in the Care and Use of Laboratory Animals.
The ADORA2A-specific agonist CGS21680 (Cat. No. 1063) and -specific inhibitor SCH58261 (Cat. No. 2270) were purchased from Tocris Bioscience (Abingdon, UK). The STAT3-specific activator Colivelin (sc-361153) and -specific inhibitor Stattic (sc-202818) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

The specific STAT3 Inhibitor V, Stattic sc-202818 (Santa Cruz Biotechnology, Catalog # 19983-44-9), was used as described before (26 (link)). A working solution of 500 µM was obtained after DMSO reconstitution and a 1/100 dilution in PBS. The CD4⁺ T cells were treated with 10 µM of the STAT3 Inhibitor V in 2 ml of complete RPMI medium (Ref R8758-500, Sigma Aldrich) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS, Ref 10270-106, Gibco), 2 mM L-glutamine, 1% streptomycin–penicillin (Ref 15140-122, Gibco), 1 mM sodium pyruvate (Ref CSTVAT00-0U, Eurobio) and 10 mM HEPES (Ref 15630-056, Gibco). A control vehicle condition was composed of allogeneic CD4⁺ cells cultured in complete DMEM medium with PBS/DMSO (final concentration of 1% DMSO). Then, allogeneic CD4⁺ T cells were stimulated with the mouse recombinant IL-10 from BioLegend (Ref #575802, Ozyme France, 78180 Montigny-le-Bretonneux) at a concentration of 100 ng/ml and incubated for 24 h at 37°C 5% CO₂. All the conditions were performed in six-plicate and the experiments were carried out twice. Cells were then collected to perform further analysis (Western blot and RT-qPCR).