Dna binding dye dapi

To assess autophagy in PBMCs stained with antibody cocktail (see Section 2.6), samples were added with dye Lysotracker Green DND-26 (Invitrogen, Eugene, OR, USA) at a concentration of 50 nM for 15 min. Cells were washed out by centrifugation in 1 mL PBS, for 5 min, at 300× g. To exclude necrotic cells from the analysis, the samples were stained with DNA-binding dye DAPI (Invitrogen, Rockford, IL, USA) at a concentration of 300 nM. Autophagy level was expressed as MFI. Anti-CD2/CD3/CD28-loaded bead treatment for 48 h was used as positive control, and autophagy inhibitor chloroquine (10 μM) was applied for the last 20 h of incubation as negative control (Supplementary Material, Figure S2).

To evaluate the expression of VCAM-1 (CD106) and ICAM-1 (CD54) adhesion molecules, HUVECs were seeded into 24-well flat-bottom plates (Sarstedt, Germany) at a concentration of 150,000 cells per ml. Then, the N2-01 in different dilutions was added for 72 h. 24 h before the end of incubation, 100 μg/ml LPS from E. coli O111:B4 (Sigma-Aldrich, United States) was added into the test wells, and the same volume of vehicle was added into the control wells. The expression of surface molecules was evaluated by flow cytometry using phycoerythrin (PE) labelled anti-CD106 (Beckman Coulter, United States, cat. No. PN A66085), anti-CD54 monoclonal antibodies (Beckman Coulter, United States, cat. № PN IM1239U) and isotype control antibody mouse IgG1-PE (Becman Coulter, United States, Cat. No PN IM0670). Single-cell suspensions staining was performed following the manufacturer's recommendation. To exclude dead cells from the analysis, the cells were stained with 1 mg/ml DNA-binding dye DAPI (Invitrogen, United States).