Phorbol 12 myristate 13 acetate

After septic stimulation for 24 h, ECs were either exposed to macrolides or to control conditions for 24 h, before washing and incubation in fresh medium. Cells were then washed three times and irradiated (20 Gy) to prevent further proliferation and cocultured with PBMCs at a ratio 1:1 for 7 days as described elsewhere.¹⁹ The supernatants of cocultures were collected after 72 h for cytokine measurement. At Day 7 of the coculture, PBMCs were stimulated by phorbol‐12‐myristate‐13‐acetate 50 ng/ml, and ionomycin 1 µM (Cell Signaling Technology) in the presence of GolgiStop (BD Biosciences) for 4 h before labeling cells to detect T lymphocytes expressing intracellular IL‐17 (CD3 ⁺CD8 ⁻IL‐17⁺) or IFN‐γ (CD3 ⁺CD8 ⁻IFN‐γ⁺) by flow cytometry²⁰, ²⁷ (Figure S1).

After incubation of ECs with IFN-γ in the presence or absence of individual or combinations of immunosuppressors, they were washed and incubated in fresh medium overnight. Cells were then irradiated (20 Gy) and cocultured with PBMCs at a ratio 1:1 for 7 days as described (42 (link)). We have previously confirmed that the irradiation step did not prevent cytokine secretion within the following 3 days (42 (link)). In indicated experiments, PBMCs from healthy donors were incubated in the presence of immunosuppressors for 24 h before coculture. The supernatants of cocultures were collected after 72 h for cytokine measurement. At the end of the coculture, PBMCs were stimulated by phorbol-12-myristate-13-acetate 50 ng/ml, and ionomycin 1 µM (Cell Signaling Technology) in the presence of GolgiStop (BD Biosciences) for 4 h before labeling cells to detect T lymphocytes expressing intracellular IL-17 (CD3⁺CD8⁻IL-17⁺) or IFN-γ (CD3⁺CD8⁻IFN-γ⁺) by flow cytometry. Carboxyfluorescein succinimidyl ester-labeled PBMCs were used to determine proliferation of Treg (CD4⁺CD45RA⁻FoxP3^bright) and Tmem (CD4⁺CD45RA⁻FoxP3^low) subpopulations.

Resveratrol (Trans-3, 4 ', 5-trihydroxystilnene), nucleosides (NS), and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, United Stated). Phorbol-12-Myristate-13-Acetate (TPA) was purchased from Cell Signaling (Danvers, MA, United States). Dulbecco’s modified Eagle’s medium (DMEM) and other culture media were obtained from Invitrogen (Carlsbad, CA, United States). A senescence associated β-galactosidase (SA-β-gal) staining kit was purchased from Cell Signaling (Danvers, MA, United States). The antibodies used for the Western blot including p53, p21, Rad9, Slug, E-cadherin, and vimentin were purchased from Genetex (San Antonio, TX, United States). Antibodies such as γ-catenin, N-cadherin, and elongation factor 1 alpha (EF1A) were purchased from Abcam (Cambridge, UK). The antibodies including β-Actin, α-tubulin, and GAPDH were purchased from Proteintech Group Inc (USA). The HRP-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Abcam (Cambridge, UK).