Ionomycin

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.

Antibodies specific for Beclin 1 phosphorylated on Ser90 were generated by Abgent (San Diego, CA, USA, 1:500). Other primary antibodies used for western blotting were anti-Beclin 1 (Cell Signaling Technology, #3738, 1:1000), anti-GAPDH (KangChen, Shanghai, China, 1:10,000), anti-p-CaMKII (Cell Signaling Technology, #3361, 1:1000), anti-CaMKII (Santa Cruz, sc-1541, 1:500), anti-Bcl-2 (Santa Cruz, SC-7382, 1:500), anti-LC3 (Novus, Littleton, CO, USA, NB100-2220, 1:3000), anti-Id-1 (Santa Cruz, sc-488, 1:1000), anti-Id-2 (Cell Signaling Technology, #3431, 1:500), anti-SQSTM1 (Santa Cruz, sc-28359, 1:1000), anti-His-Tag (Cell Signaling Technology, #2366, 1:1000), anti-Myc-Tag (Cell Signaling Technology, #2276, 1:1000), anti-Flag (Sigma-Aldrich, F1804, 1:2000), anti-ubiquitin (Santa Cruz, sc-58449, 1:1000), anti-K63-linkage-specific polyubiquitin (Cell Signaling Technology, #5621, 1:1000), anti-TRAF-6 (Cell Signaling Technology, #8028, 1:1000), anti-GAP43 (Cell Signaling Technology, #8945 s, 1:1000), anti-NF68 (Cell Signaling Technology, #2837 s, 1:1000), anti-nestin (Santa Cruz, SC-23927, 1:1000), anti-vimentin (BD, 550513, 1:1000), and anti-E-cadherin (BD, 51-9001922, 1:1000). KN-93, MG132, and bafilomycin A1 were purchased from Sigma-Aldrich. Ionomycin was purchased from Cell Signaling Technology. EB1089 was purchased from Santa Cruz.