Phloretin

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.

Myricetin, 3,4-dihydroxy phenyl ethanol, homovanillic acid sulfate, equol, genistein, phloretin, kaempferol, enterolactone, hesperetin, isorhamnetin, protocatechuic acid, trans-resveratrol, roseoflavin, pyrogallol, isoferulic acid, urolithin A, phloroglucinol, (R)-γ-valerolactone and freight were purchased from Cayman company (Ann Arbor, MI, USA). Other polyphenol standards were purified in-house through chromatographic methods with mass spectrometry/NMR confirmation. Authentic standards corresponding to the measured metabolites were purchased from Sigma-Aldrich (Saint Louis, MO, USA) or IROA Technologies (Boston, MA, USA). The stable isotope-labeled amino acid mix (20 AA U-13C, 97–99%; U-15N, 97–99%) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). HPLC-MS-grade acetonitrile, ammonium acetate, and acetic acid were all purchased from Fisher Scientific (Pittsburgh, PA, USA).

Glucose assays were performed on bioreactors and static scaffolds after 21 days cultivation to assess the rate of active transport of glucose from the apical to the basolateral sides. Bioreactors and static scaffolds were purged/washed with glucose-free 1 x Krebs buffer (126 mM NaCl, 2.5 mM KCl, 25 mM NaHCO₃, 1.2 mM MgCl₂, 2.5 mM CaCl₂) for one hour to remove any glucose from the previous media. Control (active-transport) samples were fed 2.5 mM glucose in 1 x Krebs buffer for 6 hours in the apical compartments. Passive transport samples were fed 2.5 mM glucose, 0.5 mM Phlorizin (Cayman), 1 mM Phloretin (Cayman) in 1 x Krebs buffer for 6 hours in the apical compartments. Glucose-free 1 x Krebs buffer was used in all the basolateral compartments. Samples (10 µl) were taken at time intervals from the basolateral compartments, diluted 10 fold and the concentration of glucose was assayed using Amplex® Red Glucose/Glucose Oxidase Assay Kit (Life Technologies) as per the manufacturer’s instructions.