A549 cells

RAW264.7 cells and A549 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in either arginine-free DMEM (Thermo Fisher Scientific Life Sciences, Waltham, MA, USA) or standard DMEM (containing 400 µM l-arginine; Sigma-Aldrich, St. Louis, MO, USA), both supplemented with fetal calf serum (10%; LabTech, Uckfield, United Kingdom). Confluent cultures were treated with LPS (RAW264.7 cells; 1 μg/ml; Sigma-Aldrich) or IL-1β (A549 cells; 10 ng/ml; R&D Systems, Minneapolis, MN, USA) for 24 h to induce iNOS and COX-2, in some cases in the presence of the NOS inhibitors, l-NAME (300 μM; Sigma-Aldrich) or ADMA (1 mM; Sigma-Aldrich). Increasing concentrations of ibuprofen arginate (Zambon Pharma), ibuprofen sodium (Sigma-Aldrich), l-arginine free base (Sigma-Aldrich), or the combination of ibuprofen sodium and l-arginine were also added such that the molar concentration of l-arginine present in each preparation was the same, or for ibuprofen sodium, the molar concentration of ibuprofen. After 24 h, media were collected for measurement of nitrite accumulation using the Griess reaction (Sigma-Aldrich) or PGE₂ using immunoassay (Cisbio, Codolet, France). Cell viability was assessed using the alamarBlue metabolic activity assay (Thermo Fisher Scientific).

A549 cells (American Type Culture Collection) were purchased and cultured in RPMI-1640 medium (ThermoFisher Scientific) with 10% fetal bovine serum (ThermoFisher Scientific). Then, the A549 cells were treated with 1 μM Dex or 1 μg/mL MlEVs in the presence of 10 μg/mL LPS for 24 h. IL-8 levels in culture supernatants were measured using an ELISA kit (R&D Systems), and protein expression in the cells was evaluated using the following antibodies: phospho-p65 (Cell Signaling Technology), p65 (Abcam), and actin (Santa Cruz, Dallas, TX, USA). AEC-derived EVs (AEC-EVs) were isolated by ultracentrifugation and confirmed by EV markers, including ALG-2-interacting protein X (ALIX; Santa Cruz) and tumor susceptibility 101 (TSG101; Santa Cruz). Moreover, EV protein patterns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A549 cells were purchased from ATCC (Wesel, Germany) and were cultured in DMEM F/12 medium with 10% FCS and 1% Penicillin/Streptomycin. Stimulation of A549 cells was performed after 12 h starvation (DMEM F/12 medium with 1% Penicillin/Streptomycin without FCS) with TGF-β1 (10 ng/ml, R&D Systems Inc Minneapolis, MN, USA) and TNFα (1 ng/ml, eBioscience, San Diego, USA) for the indicated time points.