Stat3 inhibitor s3i 201

DMEM (D5030), oligomycin, carbonyl cyanide 3-chorophenyl hydrazine, CCCP (Sigma # C2759), rotenone, antimycin A, STAT3 inhibitor S3I-201, and Tween 20 were purchased from Sigma-Aldrich. [U-¹⁴C]-glucose was purchased from PerkinElmer. FTY720-P CAY-10006408 was purchased from Cayman, Germany. Hoechst 33342, Rhodamine Phalloidin, MitoSOX Red (M36008), tetramethylrhodamine, ethyl ester, TMRE (T669), TrypLE™ Express Enzyme (1×), without phenol red (12604021) were purchased from Thermo Scientific, Waltham, MA, USA. The protease inhibitor mixture was Complete-Mini from Roche Basel, Switzerland, (11836153001). Immobilon-FL membranes and the phosphatase inhibitor mixture V were from Merck, Darmstadt, Germany.

Senescence-associated β-galactosidase (SA-β-Gal) activity in senescent T cells was detected as we previously described^{13 (link),14 (link)}. Naive CD4⁺ or CD8⁺ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (4.5 μM), and co-cultured with Treg or control T cells at a ratio of 4:1 in anti-CD3-coated 24-well plates for 3 or 5 days. Co-cultured naive T cells were then separated from co-cultures using fluorescence-activated cell sorting (FACS) gated on CFSE-positive populations, and then stained with SA-β-Gal staining reagent. For some experiments, the co-cultured naive T cells were determined for SA-β-Gal expression in the presence of the following inhibitors: ATM inhibitor KU55933 (10 μM, Tocris Bioscience); STAT1 inhibitor MTA (5 μM), STAT3 inhibitor S3I201 (10 μM), and JAK2 inhibitor AG490 (30 μM) (Sigma-Aldrich); MAPK inhibitors including U0126 (10 μM), SB203580 (10 μM) and SP600125 (10 μM) (Calbiochemistry). For blockade of glucose transport and glycolysis analyses, the Treg cells were pretreated with glucose transporter and glycolysis inhibitors phloretin (2 μM) or 2-deoxy-d-glucose (2-DG, 1 mM) (Cayman Chemical) for 24 h and then co-cultured with naive T cells in the presence of low concentrations of phloretin (0.5 μM) or 2-DG (300 μM) for 3 days.